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Supercoiled Protein Motifs: The Collagen Triple-Helix and the alpha-Helical Coiled Coil
Abstract
The collagen triple-helix and the alpha-helical coiled coil represent the two basic supercoiled multistranded protein motifs. Originally they were characterized in fibrous proteins, but have been found more recently in a number of other proteins containing rod-shaped domains. Coiled-coil domains are responsible for the oligomerization of proteins, as well as other specific functions, while the triple-helix domains associate to form supramolecular structures and bind a variety of ligands. Both structures were originally solved by fiber diffraction, and recent crystallographic studies on small proteins and peptide models have confirmed the structure and provided molecular details. The differences in the molecular conformations of these two motifs and the interactions stabilizing these conformations are discussed. The molecular structures of both motifs constrain the amino acid sequence to recognizable patterns, requiring the (Gly-X-Y)n repeating sequence for the collagen triple-helix and a less stringent heptad repeat requirement (h-x-x-h-x-x-x)n for the coiled-coil domains, where h represents hydrophobic residues. The features and roles of these supercoiled domains in proteins are considered when they are found adjacent in the same protein.
... The α-helix is the most common secondary structure formed when AAs wind up into a right-handed helix where the side-chains point out from the central coil. An α-helix has 3.6 residues per turn [37]. Typically, the resulting helices are stabilized via hydrogen bonding that occurs between the carbonyl oxygen of one AA and the amide proton of another AA four resides away. ...
... Supersecondary structures are intermediates between the less specific regularity of secondary structures and the highly specific folding of tertiary structures; they result from packing of adjacent secondary structure motifs, namely the α-helix and β-sheet [47]. Examples of these higher-order folding patterns include tightly wound α-helices to form coiled-coils and collagen [37], an array of β-strands to form a beta barrel [48], and combinations of multiple secondary building blocks to form protein block copolymers [49]. Although they are all assembled from the same secondary domains, these higher-ordered architectures allow proteins and peptides to perform a more diverse set of jobs compared to their individual components. ...
... Coiled-coils typically consist of two to seven right-handed α-helices that are wound around each other forming a left-handed superhelix [37]. The primary structure is amphiphilic and can be characterized by a heptad repeat (abcdefg) n . ...
Fluorination represents one of the most powerful modern design strategies to impart biomacromolecules with unique functionality, empowering them for widespread application in the biomedical realm. However, the properties of fluorinated protein materials remain unpredictable due to the heavy context-dependency of the surrounding atoms influenced by fluorine’s strong electron-withdrawing tendencies. This review aims to discern patterns and elucidate design principles governing the biochemical synthesis and rational installation of fluorine into protein and peptide sequences for diverse biomedical applications. Several case studies are presented to deconvolute the overgeneralized fluorous stabilization effect and critically examine the duplicitous nature of the resultant enhanced chemical and thermostability as it applies to use as biomimetic therapeutics, drug delivery vehicles, and bioimaging modalities.
... Further adding to the complexity is the presence of two major configurations of gelatin, the random coil and the helix, which are sensitive to temperature, pH, and solvent (32,33). These can create additional higherorder structures of coiled-helix or triple helix associations, driven by hydrophobic and hydrophilic interactions, hydrogen bonding, and presentation of side groups by the diverse range of amino acids (34,35). Gelatin random coil and helix configurations are thermoreversible, with coils dominating at elevated temperatures (>30°C) and helices at lower temperatures (34,36). ...
... These can create additional higherorder structures of coiled-helix or triple helix associations, driven by hydrophobic and hydrophilic interactions, hydrogen bonding, and presentation of side groups by the diverse range of amino acids (34,35). Gelatin random coil and helix configurations are thermoreversible, with coils dominating at elevated temperatures (>30°C) and helices at lower temperatures (34,36). As such, the swelling studies at room temperature probed the helix-dominated GelMA configuration, a higher-order structure that can lead to physical entanglements and long-range interactions that introduce increased variability compared to PVA and PEGDA systems. ...
... The initial polymer volume fraction to relaxed polymer volume fraction relationship was weakened by high variability in the relaxed polymer volume fraction data (φ r = 0.64φ 0 + 0.0206, R 2 = 0.09), and the linear fit of expected degree of polymerization between cross-links to reference ratio resulted, unexpectedly, in a negative slope ( = − 0.0056 * N c + 2.09, R 2 = 0.38). We hypothesize that the GelMA hydrogels, unlike the synthetic hydrogels whose dependence on degree of polymerization between cross-links is primarily associated with the entropic spring model, are highly affected by physical entanglements and intrachain hydrogel bonding (34,35). Therefore, reference ratio in GelMA hydrogels can be interpreted as a balance between covalent cross-links and physical entanglements (37)(38)(39). ...
Hydrogel physical properties are tuned by altering synthesis conditions such as initial polymer concentration and polymer–cross-linker stoichiometric ratios. Traditionally, differences in hydrogel synthesis schemes, such as end-linked poly(ethylene glycol) diacrylate hydrogels and cross-linked poly(vinyl alcohol) hydrogels, limit structural comparison between hydrogels. In this study, we use generalized synthesis variables for hydrogels that emphasize how changes in formulation affect the resulting network structure. We identify two independent linear correlations between these synthesis variables and swelling behavior. Analysis through recently updated swollen polymer network models suggests that synthesis-swelling correlations can be used to make a priori predictions of the stiffness and solute diffusivity characteristics of synthetic hydrogels. The same experiments and analyses performed on methacrylamide-modified gelatin hydrogels demonstrate that complex biopolymer structures disrupt the linear synthesis-swelling correlations. These studies provide insight into the control of hydrogel physical properties through structural design and can be used to implement and optimize biomedically relevant hydrogels.
... [3][4] It is also abundant in proteins forming the extracellular matrix and connective tissues. [5][6] For example, the supramolecular assembly of alpha helices of long peptide chains forms collagen fibrils in a triple helix arrangement. [5] These fibrils can also assemble into fibers to form the extracellular matrix found in all living animal tissues. ...
... [5][6] For example, the supramolecular assembly of alpha helices of long peptide chains forms collagen fibrils in a triple helix arrangement. [5] These fibrils can also assemble into fibers to form the extracellular matrix found in all living animal tissues. Coiled coils complexes are also formed transiently during biological processes such as for example the membrane fusion between presynaptic vesicles and plasma membrane which is mediated by the SNARE clamp system. ...
... In Fig. 3C is represented the evolution of the normalized adhesion energy E a =E max a with the overlapping ratio. As can be seen, E a =E max a increased gradually with a, but surprisingly, the increase displayed quantized increments (each energy increment was labeled e i with i = [1][2][3][4][5] on the figure). The total number of energy increments was equal to 5, which correlates with the number of heptads in each peptide. ...
We used the Surface Forces Apparatus to elucidate the interaction mechanism between grafted 5 heptad-long peptides engineered to spontaneously form a heterodimeric coiled-coil complex. The results demonstrated that when intimate contact between peptides is reached, binding occurs first via weakly interacting but more mobile distal heptads, suggesting an induced-fit association process. Precise control of the distance between peptide-coated surfaces allowed to quantitatively monitor the evolution of their biding energy. The binding energy of the coiled-coil complex increased in a stepwise fashion rather than monotonically with the overlapping distance, each step corresponding to the interaction between a quantized number of heptads. Surface forces data were corroborated to surface plasmon resonance measurements and molecular dynamics simulations and allowed the calculation of the energetic contribution of each heptad within the coiled-coil complex.
... Like other biological protein materials, collagen-based tissues typically have a hierarchical structure [2]. At the lowest level are polypeptide chains referred to as α-chains, which contain a repeating triplet amino acid sequence of the form Gly-X-Y, where Gly represents glycine and X and Y typically represent proline and hydroxyproline [3]. Each polypeptide chain is coiled into a left-handed triple helix and three of these α-chains coil together into a right-handed triple helix stabilised by hydrogen bonds between adjacent α-chains, resulting in a stable rod-like structure [4] (Figure 1). ...
... That is, in terms of the angles, 3 = cos ψ j−1 sin θ j−1 sin ψ j sin θ j − sin ψ j−1 sin θ j−1 cos ψ j sin θ j c j = cos ψ j−1 sin θ j−1 cos ψ j sin θ j + sin ψ j−1 sin θ j−1 sin ψ j sin θ j + cos θ j−1 cos θ j (182) ...
... !define derivatives of b_{k+1,1},b_{k+1,2},b_{k+1,3},c_{k+1} wrt th(k) for k=0,...,n-1 do k=0,n-1 dbdth(k+1,k,1) = dsin(psi(k))*dcos(th(k))*dcos(th(k+1))+dsin(th(k))*dsin(psi(k+1))*dsin(th(k+1)) dbdth(k+1,k,2) = dcos(psi(k))*dcos(th(k))*dcos(th(k+1))+dsin(th(k))*dcos(psi(k+1))*dsin(th(k+1)) dbdth(k+1,k,3) = dcos(psi(k))*dcos(th(k))*dsin(psi(k+1))*dsin(th(k+1)) & -dsin(psi(k))*dcos(th(k))*dcos(psi(k+1))*dsin(th(k+1)) dcdth(k+1,k) = dcos(psi(k))*dcos(th(k))*dcos(psi(k+1))*dsin(th(k+1)) & +dsin(psi(k))*dcos(th(k))*dsin(psi(k+1))*dsin(th(k+1)) & -dsin(th(k))*dcos(th(k+1)) enddo !define the derivative of R_{k} wrt psi(k) for k=1,...,n do k=1,n dRdpsi(k,k,1,1) = (1d0-h(k)*h(k)*b(k,1)*b(k,1))*dcdpsi(k,k)+2d0*h(k)*b(k,1)*dbdpsi(k,k,1)dRdpsi(k,k,1,2) = h(k)*b(k,1)*dbdpsi(k,k,2)+h(k)*b(k,2)*dbdpsi(k,k,1)-h(k)*h(k)*b(k,1)*b(k,2)*dcdpsi(k,k)-dbdpsi(k,k,3) dRdpsi(k,k,1,3) = h(k)*b(k,1)*dbdpsi(k,k,3)+h(k)*b(k,3)*dbdpsi(k,k,1)-h(k)*h(k)*b(k,1)*b(k,3)*dcdpsi(k,k)+dbdpsi(k,k,2) dRdpsi(k,k,2,1) = h(k)*b(k,1)*dbdpsi(k,k,2)+h(k)*b(k,2)*dbdpsi(k,k,1)-h(k)*h(k)*b(k,1)*b(k,2)*dcdpsi(k,k)+dbdpsi(k,k,3) dRdpsi(k,k,2,2) = (1d0-h(k)*h(k)*b(k,2)*b(k,2))*dcdpsi(k,k)+2d0*h(k)*b(k,2)*dbdpsi(k,k,2) dRdpsi(k,k,2,3) = h(k)*b(k,2)*dbdpsi(k,k,3)+h(k)*b(k,3)*dbdpsi(k,k,2)-h(k)*h(k)*b(k,2)*b(k,3)*dcdth(k,k)-dbdpsi(k,k,1) dRdpsi(k,k,3,1) = h(k)*b(k,1)*dbdpsi(k,k,3)+h(k)*b(k,3)*dbdpsi(k,k,1)-h(k)*h(k)*b(k,1)*b(k,3)*dcdth(k,k)-dbdpsi(k,k,2) dRdpsi(k,k,3,2) = h(k)*b(k,2)*dbdpsi(k,k,3)+h(k)*b(k,3)*dbdpsi(k,k,2)-h(k)*h(k)*b(k,2)*b(k,3)*dcdth(k,k)+dbdpsi(k,k,1)dRdpsi(k,k,3,3) = (1d0-h(k)*h(k)*b(k,3)*b(k,3))*dcdpsi(k,k)+2d0*h(k)*b(k,3)*dbdpsi(k,k,3) enddo !define the derivative of R_{k+1} wrt psi(k) for k=0,...,n-1 do k=0,n-1 dRdpsi(k+1,k,1,1) = (1d0-h(k+1)*h(k+1)*b(k+1,1)*b(k+1,1))*dcdpsi(k+1,k)+2d0*h(k+1)*b(k+1,1)*dbdpsi(k+1,k,1) dRdpsi(k+1,k,1,2) = h(k+1)*b(k+1,1)*dbdpsi(k+1,k,2)+h(k+1)*b(k+1,2)*dbdpsi(k+1,k,1) & -h(k+1)*h(k+1)*b(k+1,1)*b(k+1,2)*dcdpsi(k+1,k)-dbdpsi(k+1,k,3) dRdpsi(k+1,k,1,3) = h(k+1)*b(k+1,1)*dbdpsi(k+1,k,3)+h(k+1)*b(k+1,3)*dbdpsi(k+1,k,1) &-h(k+1)*h(k+1)*b(k+1,1)*b(k+1,3)*dcdpsi(k+1,k)+dbdpsi(k+1,k,2) dRdpsi(k+1,k,2,1) = h(k+1)*b(k+1,1)*dbdpsi(k+1,k,2)+h(k+1)*b(k+1,2)*dbdpsi(k+1,k,1) & -h(k+1)*h(k+1)*b(k+1,1)*b(k+1,2)*dcdpsi(k+1,k)+dbdpsi(k+1,k,3) dRdpsi(k+1,k,2,2) = (1d0-h(k+1)*h(k+1)*b(k+1,2)*b(k+1,2))*dcdpsi(k+1,k)+2d0*h(k+1)*b(k+1,2)*dbdpsi(k+1,k,2) dRdpsi(k+1,k,2,3) = h(k+1)*b(k+1,2)*dbdpsi(k+1,k,3)+h(k+1)*b(k+1,3)*dbdpsi(k+1,k,2) & -h(k+1)*h(k+1)*b(k+1,2)*b(k+1,3)*dcdpsi(k+1,k)-dbdpsi(k+1,k,1) dRdpsi(k+1,k,3,1) = h(k+1)*b(k+1,1)*dbdpsi(k+1,k,3)+h(k+1)*b(k+1,3)*dbdpsi(k+1,k,1) & -h(k+1)*h(k+1)*b(k+1,1)*b(k+1,3)*dcdpsi(k+1,k)-dbdpsi(k+1,k,2)dRdpsi(k+1,k,3,2) = h(k+1)*b(k+1,2)*dbdpsi(k+1,k,3)+h(k+1)*b(k+1,3)*dbdpsi(k+1,k,2) & -h(k+1)*h(k+1)*b(k+1,2)*b(k+1,3)*dcdpsi(k+1,k)+dbdpsi(k+1,k,1) dRdpsi(k+1,k,3,3) = (1d0-h(k+1)*h(k+1)*b(k+1,3)*b(k+1,3))*dcdpsi(k+1,k)+2d0*h(k+1)*b(k+1,3)*dbdpsi(k+1,k,3) enddo !define the derivative of R_{k} wrt th(k) for k=1,...,n ...
This study presents a general theory of interacting elastic curves in two dimen-sions before moving into a more general model of interacting elastic rods in three dimensions. It begins by looking at methods of modelling a single continuous two dimensional curve, before making a first attempt at modelling interactions between such curves. Finding that a discrete model would be more appropriate, the theory of two dimensional discrete curves is introduced along with a definition of discrete curvature. This model is shown to provide a good approximation to the continuous case. With promising results in the case of a single curve we model the interac¬tion between several discrete curves with great success. The general equations are then applied to a variety of interacting curve models, providing physically realistic results. Continuous space curves, curve framing, and the general theory of elastic rods is introduced in order to build discrete analogies to each, before moving on to build a discrete model of an elastic curve deforming in three dimensions. This study provides the basis to support the eventual modelling of interacting elastic rods in three dimensions. Such a model can be applied to many bundle models, including but not limited to collagen type 1 fibrils.
... A smaller peak at 42.32 • provided a d-value of 0.23 nm, which pertained to the orientation of residues present in terminal extremities (81). A small peak was observed at 54.72 • , pertaining to a dvalue of 0.168 nm, which closely matched the diameter of a single PP-II helix (82). ...
... The elevation peaked at 31.09 • representing a helical pitch of 0.279 nm, which was smaller than that of unmineralized helices (81). The peak at 55.7 • represented a length of 0.17 nm and was attributed to the radius of a single PP-II helix (82). ...
Catla collagen hydrolysate (CH) was fractionated by chromatography and each fraction was subjected to HA nucleation, with the resultant HA-fraction composites being scored based on the structural and functional group of the HA formed. The process was repeated till a single peptide with augmented HA nucleation capacity was obtained. The peptide (4.6kDa), exhibited high solubility, existed in polyproline-II conformation, and displayed a dynamic yet stable hierarchical self-assembling property. 3-D modelling of the peptide revealed multiple calcium and phosphate binding sites and a high propensity to self-assemble. Structural analysis of the peptide-HA crystals revealed characteristic diffraction planes of HA with mineralization following the (002) plane, retention of the self-assembled hierarchy of the peptide, and intense ionic interactions between carboxyl groups and calcium. The peptide-HA composite crystals were mostly of 25-40nm dimensions and displayed 79% mineralization, 92% crystallinity, 39.25% porosity, 12GPa Young's modulus and enhanced stability in physiological pH. Cells grown on peptide-HA depicted faster proliferation rates and higher levels of osteogenic markers. It was concluded that the prerequisite for HA nucleation by a peptide included: a conserved sequence with a unique charge topology allowing calcium chelation, and its ability to form a dynamic self-assembled hierarchy for crystal propagation.
... Tremendous progress has also been made in the development of artificial bones [8,9] and bone regeneration [10]. Based on our current understanding [11][12][13][14], the main components of bone have been identified as a protein collagen (mostly of type I) and an inorganic compound hydroxyapatite (HAp). The growth and maintenance of a bone are highly complex and involve many cells, proteins, hormones, ions, and, of course, water. ...
... Hydroxyapatite nanocrystals play an important role in the bone, teeth, joints, shells, and other organs of animals and belong to a class of representative systema that inspires the study and development of biogenic, biomimetic, and bionic materials, and new organicinorganic hybrid materials [22,23]. In the hierarchical structure, mineralized collagen is believed to be the most important building block and has been intensively studied, although its mass accounts for only 20 wt% to 30 wt%, while the mineral Hap, which accounts for 60 wt% to 70 wt% of the total mass, plays an auxiliary, though essential, role [11][12][13][14]. As the dominant inorganic component, the structure, morphology and dynamics of HAp still hold many unsolved puzzles. ...
Bone supports animal bodies, is the place where blood is produced, and is essential for the immune system, among other important functions. The dominant inorganic component in bone is hydroxyapatite (Hap), the structure and dynamics of which still pose many unsolved puzzles. An updated understanding of HAp is of great significance to osteology, dentistry, and the development of artificial bone and other biomaterials. In this work, HAp nanoparticles were synthesized with the wet chemical precipitation method and their structure and morphologies were controlled by varying pH and adding fluoride ions by two different routes: (1) fluoride ions were added during synthesis, and (2) fluoride ions were introduced after the samples were synthesized by soaking the samples in solutions with fluoride ions. XRD and HRTEM were employed to confirm the composition and structure, while various multinuclear (1H, 19F, 31P) solid-state nuclear magnetic resonance (NMR) methods including 1D single pulse, cross-polarization under magic-angle spinning (CPMAS), and 2D heteronuclear correlation (HETCOR) were used to characterize the structure, morphology, and dynamics, validating the general core-shell morphology in these F-HAp samples. It was found that all hydroxide ions were substituted when the fluoride ion concentration was above 0.005 M. An NMR peak corresponding to water structure emerged and the bulk water peak was shifted upfield, indicating that fluoride substitution modifies both the crystalline core and the amorphous shell of F-HAp nanoparticles. With the second route of fluoride substitution, increases in soaking time or fluoride ion concentration could increase fluoride substitution in HAp, but could not achieve complete substitution. Finally, with 1H-31P CPMAS and HETCOR, it was established that there are two types of phosphorous, one in the crystalline core (PO43−) and the other in the amorphous shell (HPO42-). These results are valuable for clarifying the fluoride substitution mechanism in HAp in biomaterials or in organisms, and provide insights for developing next generation replacement materials for bone, tooth, or coating films, drug delivery systems, etc.
... Amino acid residues of the coiled-coil motif heptad repeats are shown in upper case. The register (abcdefg) of each residue is shown in lower case, and 'a' and 'd' registers, which are critical for interfacial interactions [56,57], are underlined. B: Helical wheel representation [58] of predicted intermolecular interactions mediated by 'a' and 'd' registers of the coiled-coil motif. ...
... Coiled-coil motifs generally mediate intermolecular interactions and, less frequently, intramolecular interactions [56][57][58]. In the context of our predicted EIAV Rev structures, intermolecular coiled-coil interactions would be favored by elongated structures whereas intramolecular coiled-coil interactions would occur in globular structures. ...
Background: The lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev. Results: The tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 Å, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding.
... As described in Chapter 1, secondary structure prediction analysis of the respective amino acid sequences showed that both these proteins contain coiled-coil motifs in their N-terminal domains, suggesting that GRIF-1 and GIF 106 may constitute a novel coiled-coil domain gene family (Brickley et al., 2005a). Coiled-coil domains are important for mediating protein-protein interactions and may be involved in the homooligomersation of proteins (Beck and Brodsky, 1998). Since GRIF-1 is predicted to contain a coiled-coil domain, it was hypothesised that GRIF-1 may form homooligomers or exist as a heterooligomeric complex. ...
... These motifs have been implicated in protein-protein interactions and protein oligomerisation (Beck and Brodsky, 1998). ...
GABAA receptor interacting factor-1 (GRIF-1) is a novel protein that has been proposed to function in the trafficking of GABAA receptors and/or organelles. Evidence of in vitro association of GRIF-1 with GABAA receptor β2 subunits implicates a role of GRIF-1 in the regulation of receptor number at synapses in adult brain. GRIF-1 also shares 45% amino acid sequence identity with the human protein, β-O-linked N-acetyl glucosamine transferase (OGT) interacting protein, OIP106. Both GRIF-1 and OIP106 contain predicted coiled-coil domains in their N-terminal regions. It is thought that they constitute a novel coiled-coil domain gene family. GRJF-1 may be a species homologue of Milton, a Drosophila protein thought to function as a mitochondrial transport protein, via the microtubule-based molecular motor protein kinesin. GRIF-1 and OIP106 both interact with OGT and kinesin. Thus GRIF-1 may play a role in the anterograde transport of both GABAA receptors and/or organelles in neurons. To gain further insight into the function of GRIF-1, the quaternary structure was characterised using epitope-tagged GRIF-1 constructs in an immunoprecipitation strategy. Results showed that GRIF-1 is a soluble protein that forms disulphide-linked homo-oligomers. Size determination of recombinant GRIF-1 under native conditions using sucrose density gradient sedimentation demonstrated that GRIF-1 exists as Mr species with S20W values = 10 ± 0.8 and 13 ± 1 representing dimeric and higher molecular weight GRIF-1 complexes. The localisation of GRIF-1 was studied in primary cultures of hippocampal pyramidal neurons. Expression of exogenous GRIF-1 was investigated using an enhanced green fluorescent protein (EGFP)-tagged GRIF-1 fusion protein (EGFP-GRIF-1). Characterisation of the expression of EGFP-GRIF-1 in HEK 293 cells showed that it behaved as wild-type GRIF-1 in that it formed disulphide-linked homodimers. EGFP-GRIF-1 was transfected using Nucleofector technology. Expression of EGFP-GRIF-1 was detected over 1- 3 DIV and was colocalised with the mitochondrial marker, Mitotracker Far red 633, at the growth cones. Investigation of endogenous GRIF-1 using anti-GRIF-1 antibodies showed that endogenous GRIF-1 colocalised with the mitochondrial marker, Mitotracker™ red. Therefore GRIF-1 may play a role in the neuronal intracellular trafficking of mitochondria and/or GABAA receptors.
... The idea behind this is that similar sequences have a higher probability of forming a larger amount of hydrogen bonds, favouring a stable bond. For example, the canonical collagen motif (Gly-Pro-Pro) n easily forms hydrogen bonds to neighbouring strands if they involve the same motif because they match well in space (Brown & Timpl, 1995;Beck & Brodsky, 1998). With this knowledge, it should be possible to predict the 5 . ...
Collagens are structural proteins that are predominantly found in the extracellular matrix, where they are mainly responsible for the stability and structural integrity of various tissues. There are several different types of collagens, some of which differ significantly in form, function, and tissue specificity. Subdivisions into so-called collagen families exist, which are defined on the basis of mainly clinical research. Collagens contain polypeptide strands (ɑ-chains). Their sequences are often analysed in view of clinical aspects, but problems arise with highly homologous sequence segments. To increase the accuracy of collagen classification and prediction of their functions, the structure of these collagens and their expression in different tissues could result in a better focus on sequence segments of interest. Here, we analyse collagen families with different levels of conservation. As a result, families with strong interchain hydrogen bonds can be found, such as fibrillar collagens, network-forming collagens, and FACITs. Moreover, collagen IV α-chains form their own cluster, and other collagen α-chains do not bond at all.
... Therefore, proteases such as pepsin can degrade the polysaccharide side chains on collagen, increasing the release of C-II from the fibril structure and enhancing the extraction efficiency [17]. Removing the telopeptide region from C-II through protease cleavage can increase water solubility and potentially reduce immune responses [18]. Although undenatured C-II has oral tolerance properties, which are less than the threshold of immune response activation, preserved posttranslational modifications or structures may trigger adverse immune reactions in the human body [19,20]. ...
This study investigated whether oral supplementation with protease-soluble chicken type II collagen (PSCC-II) mitigates the progression of anterior cruciate ligament transection (ACLT)–induced osteoarthritis (OA) in rats. Eight-week-old male Wistar rats were randomly assigned to the following groups: control, sham, ACLT, group A (ACLT + pepsin-soluble collagen type II collagen (C-II) with type I collagen), group B (ACLT + Amano M–soluble C-II with type I collagen), group C (ACLT + high-dose Amano M–soluble C-II with type I collagen), and group D (ACLT + unproteolyzed C-II). Various methods were employed to analyze the knee joint: nociceptive tests, microcomputed tomography, histopathology, and immunohistochemistry. Rats treated with any form of C-II had significant reductions in pain sensitivity and cartilage degradation. Groups that received PSCC-II treatment effectively mitigated the ACLT-induced effects of OA concerning cancellous bone volume, trabecular number, and trabecular separation compared with the ACLT alone group. Furthermore, PSCC-II and unproteolyzed C-II suppressed ACLT-induced effects, such as the downregulation of C-II and upregulation of matrix metalloproteinase-13, tumor necrosis factor-α, and interleukin-1β. These results indicate that PSCC-II treatment retains the protective effects of traditional undenatured C-II and provide superior benefits for OA management. These benefits encompass pain relief, anti-inflammatory effects, and the protection of cartilage and cancellous bone.
... Collagen type I comprises about 80-90 percent of the total profile of collagen and is primarily accountable for the tendon's characteristics. The fundamental unit of collagen I consist of two 1 chains and one 2 chain (Beck and Brodsky, 1998). Along with collagen I, other small collagen forms play fundamental roles in the formation and function of tendons. ...
Tendon wounds are a worldwide health issue affecting millions of people annually. Due to the characteristics of tendons, their natural restoration is a complicated and lengthy process. With the advancement of bioengineering, biomaterials, and cell biology, a new science, tissue engineering, has developed. In this field, numerous ways have been offered. As increasingly intricate and natural structures resembling tendons are produced, the results are encouraging. This study highlights the nature of the tendon and the standard cures that have thus far been utilized. Then, a comparison is made between the many tendon tissue engineering methodologies proposed to date, concentrating on the ingredients required to gain the structures that enable appropriate tendon renewal: cells, growth factors, scaffolds, and scaffold formation methods. The analysis of all these factors enables a global understanding of the impact of each component employed in tendon restoration, thereby shedding light on potential future approaches involving the creation of novel combinations of materials, cells, designs, and bioactive molecules for the restoration of a functional tendon.
... The amide II band (1600-1500 cm − 1 ) is associated primarily with the stretching and in-plane bending vibrations of the C -N and N -H groups, respectively (Kong and Yu, 2007). The presence of quartet peaks at around 1340 and 1280, 1240, 1205 cm − 1 also serves to prove the presence of the 3-helical structure of collagen (Beck and Brodsky, 1998;Watanabe et al., 1996). ...
The aim of this study was to investigate batch-to-batch inconsistencies in the processing of pig and fish collagen isolates processed using two protocols that differed in terms of the acetic acid concentrations applied and the pre- and post-extraction steps, and which were previously tested in our laboratory with the intention of preserving the biological structures and functions of the collagen isolates for biomedical purposes. Both the major and minor components such as the amino acids, lipids, water, glycosaminoglycan and ash contents and elemental content, as well as the structure and morphology of the raw sources and the resulting batches of isolates were subsequently examined in detail applying standardized analytical methods including high perfomance liquid chromatography, ultraviolet-visible and infrared spectrometry, polyacrylamide gel electrophoresis, energy dispersive spectroscopy and scanning electron microscopy. All the fish isolates provided severalfold higher yields (8-45 wt%) than did the pig isolates (3-9 wt%). In addition, the variability of the fish isolate yields (the coefficient of variation for processing A: 16.4-32.9 % and B: 6.8-17.4 %) was significantly lower (p ≤ 0.05, n = 5) than that of the pig isolates (A: 27.7-69.8 %; B: 35.3-87.9 %). In general, the fish skin batches had significantly higher protein contents (˃60 wt%) and lower lipid contents (<10 wt%) than the pig skin batches (<55 wt% protein and up to 66 wt% lipid). In addition, the fish skin batches did not differ significantly in terms of their composition applying the same processing method, whereas the pig skin batches exhibited considerable variations in terms of their compositions, particularly regarding the protein and lipid contents. It can be stated that, concerning the fish isolates, processing B was, in most cases, slightly more efficient and reproducible than processing A. However, concerning the pig isolates, although processing A appeared to be more efficient than processing B in terms of the yield, it resulted in the production of isolates that contained a certain level of contaminants. The study provides a comprehensive discussion on the suitability of the processing protocol in terms of producing batches of reproducible quality according to the specific type of biomaterial processed from different animal species.
... The peak at 40.64 • to 40.66 • represented a distance of 0.219 to 0.22 nm, which matched with the helical rise per residue near the peptide chain extremities, particularly, non-helical regions (57). The peaks at 54.92 • to 55.11 • represented a length of 0.171 nm, which was close to the radius of a single PP-II helix (58). ...
This study attempts to identify the significant role played by the secondary and tertiary structure of collagen-derived peptides that are involved in lipid peroxide quenching in food products. Fish collagen hydrolysate (CH) was extracted with an efficiency of 70%. The constituent peptides of CH (8.2-9.7kDa) existed in a polyproline-II (PP-II) conformation and at a minimum concentration of 1mg/ml and pH range 7-8, assembled into a stable, hierarchical, quasi-fibrillar (QF) network. The peroxide quenching activity of this QF-CH increased with increasing ionic stability of the assembly and decreased upon proteolytic dismantling. Upon being used as an additive, the QF-CH reduced peroxide formation by 84.5-98.9% in both plant and fish-based oil and increased the shelf life of soya oil by a factor of 5 after 6 months of storage. The addition of QF-CH to cultured cells quenched peroxide ions generated in situ and decreased stressor activity by a factor of 12. 16 abundant peptides were identified from the CH. The reason behind the high efficacy displayed by CH was attributed to its unique charge distribution, prevalence of proton-donating amino acid residues and proximal charge delocalization by the QF network, making fish derived CH a suitable substitute for antiperoxide agents in lipid-rich food.
... These functional biomolecules can enhance the biomechanical properties of Zone I, such as its stiffness, toughness, and viscoelasticity. 49,58 In enthesis tissue engineering, the factors influencing fibrogenesis might mainly function in Zone I. ...
Enthesis, the interfacial tissue between a tendon/ligament and bone, exhibits a complex histological transition from soft to hard tissue, which significantly complicates its repair and regeneration after injury. Because traditional surgical treatments for enthesis injury are not satisfactory, tissue engineering has emerged as a strategy for improving treatment success. Rapid advances in enthesis tissue engineering have led to the development of several strategies for promoting enthesis tissue regeneration, including biological scaffolds, cells, growth factors, and biophysical modulation. In this review, we discuss recent advances in enthesis tissue engineering, particularly the use of biological scaffolds, as well as perspectives on the future directions in enthesis tissue engineering.
... α-Helix forming peptides have drawn researchers' attention through the years, as they can form nanostructures similar to those found in the cytoskeleton and the ECM of biological systems [96]. These α-helical peptides with 2-5 helices can aggregate and form nanofibers [97,98]. ...
Supramolecular peptide hydrogels have many important applications in biomedicine, including drug delivery applications for the sustained release of therapeutic molecules. Targeted and selective drug administration is often preferential to systemic drug delivery, as it can allow reduced doses and can avoid the toxicity and side-effects caused by off-target binding. New discoveries are continually being reported in this rapidly developing field. In this review, we report the latest developments in supramolecular peptide-based hydrogels for drug delivery, focusing primarily on discoveries that have been reported in the last four years (2018–present). We address clinical points, such as peptide self-assembly and drug release, mechanical properties in drug delivery, peptide functionalization, bioadhesive properties and drug delivery enhancement strategies, drug release profiles, and different hydrogel matrices for anticancer drug loading and release.
... Specifically, fibrillar collagen has approximately 340 tandem repeats of Xaa-Yaa-Gly. The amino acids in Xaa and Yaa are often occupied by Pro and 4-hydroxyproline (Hyp/O) residues, respectively, and these imino acid residues increase the thermal stability of the triple helix [2][3][4]. Isolated fibrillar collagen self-assembles into fibrils and then fibers under physiological conditions in vitro, and it then forms a gel. ...
We previously reported an artificial collagen gel that can be used as a cell-culture substrate by end-to-end cross-linking of collagen-like triple-helical peptides via disulfide bonds. However, the gel had to be formed a priori by polymerizing the peptide in an acidic solution containing dimethyl sulfoxide for several days, which prevented its use as an injectable gel or three-dimensional (3D) scaffold for cell culture. In this study, we developed a collagen-like peptide polymer by incorporating an O–N acyl migration-triggered triple helix formation mechanism into a collagen-like peptide, which formed a gel within 10 min. We demonstrated that the collagen-like peptide polymer can be used as a 3D cell scaffold and that the 3D structure formation of cells can be controlled by collagen-derived bioactive sequences introduced into the peptide sequence.
... Since G is not only the smallest amino acid but also the nonpolar, hiding it from water on the outside is energetically favorable. This constrains the amino acid sequence to the well-known pattern of GXY repeating itself axially along the triple helices [27]. Thus, the central location of the G is crucial for the stability of the collagen molecule. ...
Collagen is the most ancient molecule of the extracellular matrix as well as the most abundant protein in the body. The evolutionary conservation of collagen can be seen from its wide utilization in many different tissues throughout the animal kingdom. The collagen molecule has a triple-helical structure assembled by cells through a complex multistep and elaborate process, the collagen biosynthesis. The helical structure is reinforced by a network of hydrogen bonds that significantly enhance the thermal stability as well as contribute to the elasticity of the molecule. This chapter provides an overview of the structure, evolution, biosynthesis, and structure-function relationships of the collagen molecule and discusses polymer- and molecular dynamics-based models of elasticity and how they predict changes in stiffness following point mutations in the primary protein structure of collagen that occur in osteogenesis imperfecta.
... From the results, it could be referred that the thickness of the monolayer gelatin molecule was about 4-6Å, which was similar to previous works. [40][41][42] When the addition of gelatin was less than 0.75%, the d-value might be mainly governed by a monolayer arrangement of the gelatin molecules. Concentration beyond 0.75% and up to 1.5% caused a linear increase of the d-value. ...
In this paper, sodium montmorillonite was modified with gelatin of different concentrations, and various colloidal characteristics of the gelatin-treated clays were measured and analyzed in detail. First, the influence of gelatin on the interlayer space of Mt layers was investigated by X-ray diffraction analysis. Moreover, the aggregation of Mt particles was examined using a combination of electron microscopy and particle size distribution experiments, while the variation of the electrical property of Mt was measured using ζ potential test. Gelatin of different concentrations can increase the particle size of Mt in different degrees. The addition of 4% gelatin could improve the ζ potential of Mt from −30.65 to −15.55 mV. The wettability change of modified Mt induced by the adsorption of gelatin was followed by measurements of water contact angle and observations of the morphology of Mt/gelatin membrane through SEM images. 4% gelatin could improve the water contact angle of Mt to 81.3°. Finally, the rheological properties of Mt/gelatin dispersion including shear viscosity and shear stress were measured using a stress-controlled rheometer. All of the results were consistent by showing that the overall colloidal characteristics and behavior of the gelatin-treated Mt strongly varied depending on the gelatin concentration used in the modification process. These results can provide a deep and comprehensive understanding of the colloidal properties of clay/gelatin systems and give important guidance for the performance design and improvement of Mt/gelatin composite materials. Furthermore, this study can also be expanded the application of gelatin and its composites to other fields.
... Sticky-end assembly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 A c c e p t e d M a n u s c r i p t 13 thus reduces the heterogeneity of a heterotrimer mixture. 74 For instance, the Hartgerink group has improved the self-assembly of CLPs via charge-pair 67 and amide-π 75 interactions between residues in the "axial" and "lateral" positions on adjacent strands ( Figure 3C, Table 1). Overall CLPs represent a versatile class of compounds useful for both targeted drug delivery and for use as mechanical scaffolds. ...
Proteins are some of the most versatile and studied macromolecules with extensive biomedical applications. The natural and biological origin of proteins offer such materials several advantages over their synthetic counterparts, such as innate bioactivity, recognition by cells and reduced immunogenic potential. Furthermore, proteins can be easily functionalized by altering their primary amino acid sequence and can often be further self-assembled into higher order structures either spontaneously or under specific environmental conditions. This review will feature the recent advances in protein-based biomaterials in the delivery of therapeutic cargo such as small molecules, genetic material, proteins, and cells. First, we will discuss the ways in which secondary structural motifs, the building blocks of more complex proteins, have unique properties that enable them to be useful for therapeutic delivery. Next, supramolecular assemblies, such as fibers, nanoparticles, and hydrogels, made from these building blocks that are engineered to behave in a cohesive manner, are discussed. Finally, we will cover additional modifications to protein materials that impart environmental responsiveness to materials. This includes the emerging field of protein molecular robots, and relatedly, protein-based theranostic materials that combine therapeutic potential with modern imaging modalities, including near-infrared fluorescence spectroscopy (NIRF), single-photo emission computed tomography/computed tomography (SPECT/CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound/photoacoustic imaging (US/PAI).
... Natural collagen is a rod-like macromolecule with a length of ca. 280 nm and a diameter of 1-2 nm, consisting of three parallel polyproline type II helices with unique repeats of X aa -Y aa -Gly (X aa and Y aa are Pro and Hyp in the most frequent triplet), and enables the formation of microscale-ordered fibrils [20][21][22]. In synthetic systems, triple-helix-based nanofibers have also been constructed from short CMPs by incorporating a "sticky-end" strategy [23,24] or specific assembly signals into both ends of a CMP triple helix, such as electrostatic interaction [25][26][27], π-π stacking [28,29], π-cation interaction [30,31] and metal-ligand coordination [32,33] to permit linear growth. ...
Self-assembly of artificial peptides has been widely studied for constructing nanostructured materials, with numerous potential applications in the nanobiotechnology field. Herein, we report the synthesis and hierarchical self-assembly of collagen-mimetic peptides (CMPs) bearing various aromatic groups at the N-termini, including 2-naphthyl, 1-naphtyl, anthracenyl, and pyrenyl groups, into nanofibers. The CMPs (R-(GPO)
n
: n > 4) formed a triple helix structure in water at 4 °C, as confirmed via CD analyses, and their conformations were more stable with increasing hydrophobicity of the terminal aromatic group and peptide chain length. The resulting pre-organized triple helical CMPs showed diverse self-assembly into highly ordered nanofibers, reflecting their slight differences in hydrophobic/hydrophilic balance and configuration of aromatic templates. TEM analysis demonstrated that 2Np-CMP
n
(n = 6 and 7) and Py-CMP
6
provided well-developed natural collagen-like nanofibers and An-CMP
n
(n = 5-7) self-assembled into rod-like micelle fibers. On the other hand, 2Np-CMP
5
and 1Np-CMP
6
were unable to form nanofibers under the same conditions. Furthermore, the Py-CMP
6
nanofiber was found to encapsulate a guest hydrophobic molecule, Nile red, and exhibited unique emission behavior based on the specific nanostructure. In addition to the ability of CMPs to bind small molecules, their controlled self-assembly enables their versatile utilization in drug delivery and wavelength-conversion nanomaterials.
... Each of these proteins contains predicted coiled-coil motifs in their N-terminal domains, shown in Figure 5.1. Coiledcoil domains are found in many proteins throughout the cell and in the extracellular matrix and are important for mediating protein-protein interactions (Beck and Brodsky, 1998). The GABAa receptor p2-IL interacting domain of GRIF-1 was localised to residues 124-283. ...
γ-Aminobutyric acid type A (GABAA) receptors are the most abundant inhibitory neurotransmitter receptors in the mammahan central nervous system. They are ligand- gated chloride ion channels. Each receptor is composed from 5 of the 16 known subunits αl-6, βl-3, δ, γ1-3,δ,π, &thetas; and s. Each subunit type confers different properties to the fully assembled receptor, leading to a diverse range of possible receptor subtypes. However, very few of the theoretically possible subtypes are actually observed in vivo. Here, a series of studies has been undertaken, using the yeast two-hybrid system, to investigate various aspects of GABAA receptor assembly and trafficking, to understand the molecular basis of the observed receptor diversity. Firstly, since GABAA receptor N-terminal domains have been implicated in subunit associations, α1 and β2 subunit N- termini were studied to identify assembly motifs, using 3 different yeast two-hybrid systems, the GAL4, modified LexA and CytoTrap® systems. Secondly, trafficking of receptors and the development and stabilisation of GABAergic synapses were investigated by screening a rat brain cDNA library using the GABAA receptor β3 subunit intracellular loop (β3-IL) and β2 N-terminal domain, respectively, to identify novel interacting proteins. The p2 N-terminus was found to interact with a DnaJ- domain-containing sequence, TIDIL. Thirdly, receptor trafficking was investigated by further characterisation of the previously identified novel protein, GABAA receptor interacting factor (GRIF-1). The binding specificity of GRIF-1 with the GABAA receptor β2-IL was analysed. Structural and functional similarities between GRIF-1 and other members of the novel coiled-coil domain-containing gene family of proteins were investigated. GRIF-1 and a human homologue, KIAA1042, were compared for interactions with the GABAA receptor β2-IL and with kinesin heavy chain (KHC), KIF5C, as a KHC has been shown to associate with Milton, the Drosophila melanogaster orthologue of GRIF-1. Despite the high degree of amino acid homology between GRIF-1 and KIAA1042, only GRIF-1 was found to bind to the GABAA receptorβ2-IL and to KIF5C, suggesting that the subtle differences between GRIF-1 and KIAA1042 lead to differences in functional specificity.
... The peaks at 54.92-55.11° represented a length of 0.171nm, which was close to the radius of a single PP-II helix [49]. ...
This study attempts to identify the significant role played by the secondary structure of collagen-derived peptides that are involved in lipid peroxide quenching in food products. Collagen was extracted from the skin of Perch and swim bladder of Rohu at 45-78% efficiency. It was identified as type-I based on a high molecular weight (110kDa) and its ion-exchange elution profile. The collagen samples were enzymatically hydrolyzed and collagen hydrolysate (CH) was extracted with an efficiency of 0.67-0.74g/g of collagen. The CH samples displayed a molecular weight in the range of 8.2-9.7kDa and exhibited a higher abundance of charges resulting in higher solubility. The structural studies revealed that the CH peptides existed in polyproline-II helix and formed a mimic-triple helix in a wide range of pH. In neutral and alkaline pH, the mimic helices joined to form a hierarchical quasi-fibrillar network that was smaller than collagen fibrils but also more dynamic. The CH exhibited >95% degradation in 15h through simulated digestion. The CH were able to decrease peroxide formation by 84.5-98.9% in commercially available cod liver and almond oil and increased the shelf life of soya bean oil by a factor of 5 after 6 months of storage. The addition of CH to cultured cells quenched peroxide ions generated in situ and decreased stressor activity by a factor of 12. The reason behind the high efficacy of CH was deciphered to be the proximal charge stabilization by the quasi-fibrillar network, which allowed efficient peroxide quenching and long-term stability.
... Those proteins feature various structural organizations. In all cases, one or two collagenous segments are combined to other domains such as phage fiber domains and alpha coiled coil domains that were found to be trimerisation domains 189 . This protein building plan leads to formation of long flexible rods connecting globular domains that were proposed to form side fibers in the tails of bacteriophages 159 . ...
Gp12 is a protein found distributed symmetrically at the surface of the icosahedral capsid from bacteriophage SPP1. Recombinant gp12 binds to phage particles whose gene coding for gp12 was disrupted. This interaction occurs specifically with capsids that undergone expansion and packaged DNA.The gp12 protein sequence is marked by the presence of a stretch of 8 repeats of a GXY motif, which is the sequence signature of collagen. Our results showed that gp12 is an elongated trimer in solution. The trimer is sensitive to collagenase VII that cuts the gp12 protein inside the collagen motif. Its circular dichroism profile has also the signature of a collagen-like protein. Binding of gp12 to SPP1 capsids increases its thermal stability by 20°C. Gp12 is denatured and dissociated reversibly by temperature shift. The gp12 trimer and its denatured form bind to SPP1 capsids but with a different interaction behavior. These properties allow to use gp12 as thermo-switchable SPP1 capsid binder. Gp12 has a modular organization with a central collagen motif that connects the amino and carboxyl termini. Proteins with a similar organization that are encoded by genes adjacent to the gene coding for the major capsid protein were identified in prophages of Bacilli, suggesting a function similar to gp12. Their modules have a variable length.A pangenome-wide search for collagen-like proteins in prokaryotes and viruses shows that they are abundant among bacteria and viruses. In contrast, this motif is rare is archaea and their viruses. Our analysis highlights the importance of collagen-like proteins in the non-eukaryotic world and supports the interest to develop their biochemical and structural study.
... Chirality is present in collagen at all structural scales: peptide units are L-amino acids, tropocollagen molecules are composed of three left-handed α chains wound together as a right-handed triple helix, and fibrils show a left-handed helical twist [41][42][43]. Collagen thus exhibits optical activity, notably circular dichroism (CD), which is theoretically accounted for by the introduction of magnetic dipolar and electric quadrupolar terms [31]. CD-SHG is the nonlinear analog of conventional (linear) CD. ...
... Proline is often represented by X and hydroxyproline is by Y, respectively. 10 The glycine, proline, and hydroxyproline account 50% of the total constituent of the amino acids in fibrillar collagen, which are responsible for providing the triple-helical strength and stability. 11 The collagen fibers become less soluble, thinner, shortened, and disorganized due to skin aging. ...
Background
Skin aging is a dynamic process that affects the entire body, marked by molecular and structural changes. Type I collagen is the most abundant structural component and accounts 80% of total collagen in human skin. The amount of proline and hydroxyproline reflect the quantity and quality of the collagen fiber in the extracellular matrix of skin, which is alerted due to accumulated effects of intrinsic and extrinsic aging. Extrinsic aging is driven by ultraviolet radiation–induced reactive oxygen species production that activates the matrix metalloproteinase and disrupts the extracellular matrix of skin dermis, while intrinsic aging is the non‐enzymatic process resulting in advanced glycation end products (AGEs). In the presence of pentosidine‐AGEs, aging process is accelerated.
Method
In vivo Raman spectra of human dermis were collected from forearms of 30 volunteers and were divided into three groups: 10 young adult 25 ± 5 years, 10 old adult 65 ± 10 years and 10 diabetic old adult 65 ± 10 years old male participants. Density functional theory was performed to compute the vibration modes of AGEs, pentosidine, and glucosepane.
Results
In vivo confocal Raman spectroscopy detects the specific changes in the proline and hydroxyproline conformation, collagen fiber degradation of type I collagen and AGE protein contribution to specific Raman bands in the aged dermis because of Intrinsic and Extrinsic aging. Statistical t test marked significant differences (P < .01) in Raman peaks of proline and hydroxyproline among young adult, old adult, and diabetic old adult participants at wavenumbers 855, 875, 922, and 938 cm⁻¹.
Conclusion
In vivo confocal Raman spectroscopy is a useful tool to detect the AGE markers in the old adult and diabetic old adult male participants, which interacts with the ultraviolet radiations and accelerates the aging process resulting in the extracellular matrix degradation.
... Chirality is present in collagen at all structural scales: peptide units are L-amino acids, tropocollagen molecules are composed of three left-handed α chains wound together as a right-handed triple helix, and fibrils show a left-handed helical twist [41][42][43]. Collagen thus exhibits optical activity, notably circular dichroism (CD), which is theoretically accounted for by the introduction of magnetic dipolar and electric quadrupolar terms [31]. CD-SHG is the nonlinear analog of conventional (linear) CD. ...
Second-harmonic generation (SHG) microscopy is currently the preferred technique for visualizing collagen in intact tissues, but the usual implementations struggle to reveal collagen fibrils oriented out of the imaging plane. Recently, an advanced SHG modality, circular dichroism SHG (CD-SHG), has been proposed to specifically highlight out-of-plane fibrils. In this study, we present a theoretical analysis of CD-SHG signals that goes beyond the electric dipolar approximation to account for collagen chirality. We demonstrate that magnetic dipolar contributions are necessary to analyze CD-SHG images of human cornea sections and other collagen-rich samples. We show that the sign of CD-SHG signals does not reveal whether collagen fibrils point upwards or downwards as tentatively proposed previously. CD-SHG instead probes the polarity distribution of out-of-plane fibril assemblies at submicrometer scale, namely homogeneous polarity versus a mix of antiparallel fibrils. This makes CD-SHG a powerful tool for characterizing collagen organization in tissues, specifically the degree of disorder, which is affected during pathological remodeling. CD-SHG may thus serve to discriminate healthy and diseased collagen-rich tissues.
... An analysis of the predicted amino sequence (635 amino acids from the 2.2 kb of nucleotide sequence) of C3 using PC GENE and the Sanger centre prosite program (www.sanger.ac.uk), which searches for fimctional domains within amino acid sequences. Brodsky, 1998). The recently identified protein, yotiao, which is predicted to be important in the anchoring of NMDA receptors to the postsynaptic cytoskeleton, has an extensive coiled-coil domain structure. ...
γ-Aminobutyric acidA (GABAA) receptors are members of the ligand-gated ion channel superfamily. They are hetero-oligomeric proteins which are composed of varying combinations of subunits to form functional receptors. The fifteen known GABAA receptor genes code for multiple subunit classes which are categorised by their respective amino acid sequence homologies i.e. α1-α6, β1-β3, γ1-γ3, δ, ε and π. The anchoring and clustering of neurotransmitter receptors at positions apposed to the site of neurotransmitter release is important for the efficient functioning of chemical synapses. The interaction of specific proteins with neurotransmitter receptors has been demonstrated to be necessary for this anchoring and clustering process. We therefore used the yeast two-hybrid system to identify potential GABAA receptor anchoring proteins. The DNA encoding the GABAA receptor β2 intracellular loop was used as the bait to screen a rat brain cDNA library of 1.8×10^6 recombinants. Three clones encoding protein sequences which interact with the intracellular loop of the GABAA receptor β2 subunit have been identified. One of these contained an insert of 1.9 kb which database searches have shown to be a novel sequence. The searches did however show a 84% DNA identity to the human expressed sequence tag (EST) zm75b03.rl. Northern blot analysis showed that the clone hybridised with three transcript sizes of 6.2 kb, 4.2 kb and 2.9 kb. The larger transcript was expressed in heart, brain, spleen, lung, liver, skeletal muscle and kidney but not in testis. The smallest transcript was expressed primarily in the testis and to a much lesser extent in the heart, brain, spleen, liver, lung, skeletal muscle and kidney. The 1.9 kb clone was radiolabelled and used as a probe to identify other hybridising clones enabling the determination of further nucleotide sequence. The creation of a GST fusion protein of the GABAA receptor β2 intracellular loop and a polyHisCS fusion protein allowed antibodies to C3 to be produced and 'pull-down' assays to be performed to further characterise the interaction between C3 and the GABAA receptor β2 intracellular loop.
... Chitin nanofibrils of pens have been characterized as being surrounded by proteins with helical and coiled-coil structures (Yang et al., 2014). We identified seven proteins embedded in the pen with these properties including collagen, which is a supercoiled, triple helix (Beck and Brodsky, 1998). We also found other proteins which have α-helical secondary structures including ADAMTS (Gerhardt et al., 2007), chitinase ( Hahn et al., 2000), chitin deacetylase ( Liu et al., 2017), proteins with Kunitz domains (Dai et al., 2012) and omega crystallin (Vasiliou et al., 2013). ...
The pen or gladius of the squid is an internalized shell and serves as a site of attachment for important muscle groups and as a protective barrier for the visceral organs. The pen's durability and flexibility is derived from its unique composition of chitin and protein. We report the characterization of the structure, development, and composition of pens from Doryteuthis pealeii. The nanofibrils of the polysaccharide, β-chitin, are arranged in an aligned configuration in only specific regions of the pen. Chitin is secreted early in development enabling us to characterize the changes in pen morphology prior to hatching. The chitin and proteins are assembled in the shell sac surrounded by fluid which has a significantly different ionic composition than squid plasma. Two groups of proteins are associated with the pen, those on its surface and those embedded within the pen. Only twenty proteins are identified as embedded within the pen. Embedded proteins are classified into 6 groups including chitin associated, protease, protease inhibitors, intracellular, extracellular matrix and those that are unknown. The pen proteins share many conserved domains with proteins from other chitinous structures. We conclude that the pen is one of the least complex, load-bearing, chitin-rich structures currently known, and is amenable to further studies to elucidate natural construction mechanisms using chitin and protein. 3
... dϕ denotes mean value on the part of each fibril f of pitch P that is contained in the transverse PSF. Indeed, for skin and vessels, the pitch P is about 1 µm [22,62], which is greater than the size of the transverse PSF (w T = 0.4µm, see section Materials and methods) such that ∆ϕ = 2π × w T /P. For tendon, P∼20 µm is estimated from the SHG images by the average distance between two maximums along each fibril (see Fig. 3(a)). ...
From P-SHG experiments, second-order nonlinear optical anisotropy parameters ρ = χ ZZZ /χ ZXX of collagen tissues are calculated assuming the same model of supercoiled collagen fibril characterized by a variable angle θ. Dispersion of experimental ρ values is converted into distribution of θ values based on the wavy nature of collagen fibrils deduced from EM studies. For tendon, the results show that the dispersion of experimental ρ values is mainly due to Poisson photonic shot noise assuming a slight fibrillar undulation with θ = 2.2° ± 1.8°. However for skin and vessels, the dispersion of experimental ρ values is mainly due to a stronger fibrillar undulation with θ = 16.2° ± 1.3°. The results highlight that this undulation is reduced during the development of liver fibrosis therefore, contributing to the rigidity of the tissue.
... BRP and CAST1/ERC2 share structural similarities mainly in the N-terminal domain [11] suggesting that CAST1/ERC2 could be a mammalian counterpart of BRP, with both of them sharing coiled-coil (CC) domains. CC domains are α-helical structures that self-assemble promoting the nucleation of large molecular complexes, resulting in the formation of multimeric and homomeric assemblies [15][16][17]. An interesting characteristic of CAST1/ERC2 is its propensity to form aggregates when expressed in heterologous cells. ...
Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation. The self-assembly and synaptic recruitment of the Drosophila protein Bruchpilot (BRP), a partial homolog of CAST1/ERC2, is modulated by the serine-arginine protein kinase (SRPK79D). Here, we demonstrate that overexpression of the vertebrate SRPK2 regulates the self-assembly of CAST1/ERC2 in HEK293T, SH-SY5Y and HT-22 cells and the CC1 and CC4 domains are involved in this process. Moreover, the isoform SRPK2 forms a complex with CAST1/ERC2 when co-expressed in HEK293T and SH-SY5Y cells. More importantly, SRPK2 is present in brain synaptic fractions and synapses, suggesting that this protein kinase might control the level of self-aggregation of CAST1/ERC2 in synapses, and thereby modulate presynaptic assembly.
... The Gly-X-Y motif is a collagen-like repeat composed of a glycine residue that recurs in every three amino acid, followed by the positions X and Y, which are often filled by proline residues (Beck and Brodsky, 1998). The glycine and proline residues confer a stable triple helical structure to the mammalian collagen. ...
Gly-X-Y (Gly represents glycine) motif is a collagen-like repeat present at the N-terminal domain of bacteriophage hyaluronate lyase (Phage HL). It addition in providing conformational stability to the protein, it also has non-specific regulatory role in substrate binding as well as in modulation of the enzymatic activity in vitro. The presence of Gly-X-Y motif in the prophage genome is a virulence factor of vertebrate origin that arose through lateral gene transfer. On the basis of a number of evidences we hypothesise that the Gly-X-Y repeat might help the Phage HL in spreading infection by adhering at the surface of the human epithelial cell and assist in congregation of phage particles at the site of infection. The Gly-X-Y repeat may also cause polyarthritis by cross-reacting with tissue collagen during the infection of host streptococci. Therefore we propose that group A streptococcus may exploit the Phage HL for the possible pathogenesis by using the cell adherence property of Gly-X-Y collagen repeats. This is a new avenue of research in Phage HLs as the Phage HL has broad substrate degradation propensity.
... 33 Average spectrum suggests major changes in the overall collagen content of the tissue, Fig. 2. Origin of Raman band around 856 cm −1 has been assigned to the proline ring in the collagen polypeptide chain. 27,28,34 The band around 1450 cm −1 originates from CH 2 bending deformation of collagen, protein, and lipids. Enhanced intensity of these bands in the LPS treated samples indicate higher collagen content. ...
The tympanic membrane (TM) is a dynamic structure that separates the middle ear from the external auditory canal. It is also integral for the transmission of sound waves. In this study, we demonstrate the feasibility of using Raman spectroscopy to identify early chemical changes resulting from inflammation in the TM that can serve as an indicator of acute otitis media. Bacterial lipopolysaccharide (LPS) was injected trans-tympanicaly in a murine model. Presence of inflammatory response was assessed with binocular microscopy, confirmed with histopathology and immunofluorescence staining. Successful discrimination suggesting spectral differences among the control and LPS treated groups was achieved using principal component analysis. Raman imaging revealed major differences in collagen distribution and nucleic acid content. Image segmentation analysis on the trichrome stained tissue sections was performed to corroborate the Raman spectra. The spectral co-localization study suggests changes in the expression of collagen IV specific signals in LPS treated samples. The overall findings of the study support prospective application of RS in the diagnosis and therapeutic monitoring of otitis media.
Majority of biomimetic implant coatings display high deviation in component HAp crystal sizes from the Goldilocks range of 25-50nm resulting in low osseointegrative potential, consequently leading to delay in implant acceptance and, in extreme situations, complete rejection. The present study attempts to use a novel peptide to crystallize HAp coating with a higher percentage of crystals in the desired Goldilocks zone, ideally leading to faster osteoblast-implant surface bonding. Collagen hydrolysate was extracted from fish scales and a novel HAp-nucleating peptide (PF) was isolated by sequential chromatography. It displayed a widely conserved sequence with a PP-II conformation that further coiled to form triple helices. At physiological pH, these triple helices self-assembled into a quasi-fibrillar network (QFN) with dimensions of 842.2±229nm. The HAp synthesized on PF-QFN (PFC) displayed lower N1s binding energy in XPS indicating greater dynamicity, while an augmented Ca2p bond in XPS and intense FTIR bands at 1384cm-1 revealed strong carboxyl-calcium interaction, which, coupled with a prominent (002) plane along the QFN-axis, culminated in augmented crystallinity (89.2%). Consequently, PFC crystals were 29% harder (162 MPa) with a 75% elevated Young’s modulus (4.7GPa) when compared to currently used collagen-based composites (CLC). The average PFC crystal dimensions were 48 × 28 nm2, which, upon being used as a coating, was 98% anti-bacterial, 94% higher corrosion-resistant, and 15% more osteoinductive with an average 25% increase in osteogenic markers when compared to present biomimetic coatings. Overall, the results established that the PF based nanocomposite was a suitable agent for implant and bone graft coating material.
Significant efforts have been dedicated to the design of protein and peptide-based nanomaterials using a bottom-up approach with potential utility in biomedical and nanotechnology fields. Artificial peptides are fascinating building units because of their structural and functional diversity, easy synthesis, and biocompatibility. This review describes a hierarchical self-assembly of amphiphilic collagen-mimetic peptides (CMPs) bearing various aromatic groups at the N-termini into various nanostructures, such as nanofibers, vesicles, and micelle fibers, as well as their stimuli-responsiveness and fluorescence behavior. The relationship between CMP structure and the consequent morphology of self-assembly is discussed in detail.
L’asthme allergique sévère est une pathologie inflammatoire chronique conséquence d’uneréponse inappropriée de l’organisme à un l’allergène. Cette pathologie, qui représente unproblème de santé publique, peut se caractériser par un infiltrat pulmonaire à neutrophiles,associé à un remodelage bronchique accentué, souvent résistant aux thérapeutiqueshabituelles (corticoïdes inhalés), et à une expression de cytokines de type Th17, menant à un profil Th2/Th17. Les cytokines Th17 comme l’IL-17 et l’IL-22 sont trouvées dans les expectorations induites de patients asthmatiques et ont des propriétés pro-inflammatoires dans le cas de l’asthme allergique. L’IL-22 est impliquée dans le recrutement de neutrophiles,dans le remodelage tissulaire et est connue pour être induite suite à l’infection par des pathogènes de l’enfance qui contribuent au développement et aux exacerbations de l’asthme.Ces pathogènes sont reconnus par un récepteur intracellulaire de l’immunité innée, NOD2,présent au niveau des cellules épithéliales et dendritiques. Ce récepteur est capable de reconnaitre un peptidoglycane, le muramyl dipeptide. Un effet synergique pourrait ainsi existerentre des éléments environnementaux que sont les allergènes et différents agents infectieuxaux quels nous sommes confrontés dans la petite enfance qui pourraient avoir un effet adjuvant sur la production d’IL-22. Pour répondre à cela, un modèle murin d’asthme chronique neutrophilique à l’allergène de chien a été développé au laboratoire, reproduisant l’asthme sévère d’endotype Th2/Th17. La première partie du projet a évalué les effets de la costimulation par NOD2 dans ce modèle chronique, en particulier le remodelage des voies aériennes et l’origine cellulaire de la production d’IL-22. Si l’hypothèse testée d’un effet adjuvant de NOD2 sur les paramètres de l’asthme sévère n’a pas été confirmée, la formation d’inductible Tissu Lymphoïde Associé aux Bronches (iBALT) dans ce modèle nous a faitpoursuivre la caractérisation phénotypique et mécanistique de la formation de ces follicules.Dans ce contexte, l’implication d’un facteur de transcription, exprimé par les cellules Th17 etessentiel à la production d’IL-22, l’Aryl hydrocarbon receptor (AhR) a été évalué. Dans notre modèle, un antagoniste de l’AhR inhibe certains paramètres de l’asthme sévère dont la production d’IL-22, mais à l’inverse, va augmenter la formation des iBALTs en diminuant légèrement la production d’IL-10. La seconde partie a pour but de déterminer le rôle de l’IL-22dans ce modèle chronique d’asthme expérimental à l’allergène de chien, par l’utilisation de souris déficientes en IL-22. Nous observons que l’absence d’IL-22 atténue certaines réponses associées à l’asthme dont, le recrutement de neutrophiles, l’hyperréactivité bronchique, les paramètres liés à la formation de iBALTs, mais ne modifie pas la réponse humorale. La dernière partie de ce projet, vise à identifier les cellules cibles impliquées dans les effets dépendant de l’IL-22, grâce à des souris déficientes en IL-22Ra1, une sous-unité du récepteur de l’IL-22 exprimé, notamment, sur les cellules épithéliales ou les cellules musculaires lisses/myofibroblastes. Les premiers résultats montrent un effet des deux types cellulaires surle recrutement cellulaire global, et des cellules musculaires lisses pour la résistance des voies aériennes et la réponse humorale.En conclusion, nos résultats indiquent que l’IL-22 est une cible de choix dans le cadre d’un traitement de l’asthme allergique sévère. L’ensemble de ce projet devrait permettre la mise en évidence de nouveaux mécanismes impliqués dans l’asthme sévère et ouvrir la voie vers de nouvelles stratégies thérapeutiques pulmonaires locales, ciblant l’IL-22Ra1 et potentiellement le remodelage bronchique. Un des indicateurs de sévérité de l’asthme mais pour lequel aucun traitement n’est disponible en dehors de la thermoplastie.
Edible packaging has long been used in food preservation as casing, coating and self-standing film. Nearly 4000-year-old Sumerian tablets, discovered in Mesopotamia, suggest that small or large intestines of cattle or sheep had been used as natural casing for sausages since ancient times. The first-time use of edible coatings in fruits is attributed to the Chinese, who formulated and applied edible wax coatings for preservation of oranges in the 12th century. Moreover, it is also thought that yuba, a proteic film that formed on the surface of boiled soy milk, was used by the Japanese in the 15th century as the first self-standing edible film applied for wrapping food. Although records about systematic use of ‘active edible packaging in ancient times are scarce, it should be kept in mind that the ancient process of smoking applied to sausages yields an antimicrobial edible film, causing accumulation of antimicrobial smoke components (e.g. acids, phenol, carbonyl) within the casing. However, the patents of 1950s, related to incorporation of antifungal food preservatives into edible pectin films, suggest that scientists became aware of the potential benefits of active edible packaging in the middle of 20th century. This chapter focuses on the definition of active edible packaging and its major concepts, such as antimicrobial, antioxidant, flavor-release, and bioactive packaging. The potential of different hydrocolloids to form edible packaging (e.g. film, coating, casing, nanofiber mat, etc.) has also been discussed briefly to prepare readers for the following chapters.
Precise control over the smart materials exhibiting reversible shape changes in response to environmental stimuli presents a considerable challenge. Here, with a self-assembly strategy by extracting natural materials from pigskin, a single layer bio-inspired, transparent, soft biological film (BF) with the primary characteristics of self-actuation and self-sensing is successfully developed. The self-assembly constructed BF can exchange water and reflect environmental humidity gradients rapidly to activate continuous rotary movement. Temperature which affects the thermal motion of water molecules will induce different orientation movement of the film, and on this basis, a humidity-driven energy transfer motor is developed. More characterizations highlight the behavior mechanism of BF through water exchanging by a hydrogen bonding interaction with the hydrophilic group of amino acids residues on the BF surface. Finally, a wearable, steady and ultrafast-response sensor to detect human breathing, especially for real-time obstructive sleep apnea (OSA) state, is fabricated. This study offers great potential in emerging applications including micro-sensors, switches, soft robots and power source technologies.
The book reviews the potential of natural hydrocolloids and active agents to develop sustainable edible packaging materials for food preservation. Natural hydrocolloids have been covered including their current and future sources, extraction methods, health effects (both positive and negative), and their ability to form different types of packaging such as film, casing, coating, mat, pad, etc. Similarly, natural active compounds have been evaluated carefully considering their sources, extraction methods, regulatory status, and compatibility with edible packaging. The book lays particular emphasis on recent developments in methods, strategies and technologies employed to enhance the performance of antimicrobial, antioxidant and bioactive packaging. The basic testing methods used to evaluate antimicrobial and antioxidant activity of edible packaging in model media and food have been discussed, and carefully selected example active edible packaging applications for different food categories have been provided with critical details such as the thin balance between effectiveness of packaging and sensory properties of food. The book helps in understanding necessary parameters in designing a natural active edible packaging that is applicable to a specific target category of food. Moreover, readers are primed, for the first time, on how to develop a high performance, but fully natural active edible food packaging.
This book is different from most similar books as it provides neither methodologies about classical active packaging based on chemicals and fossil polymeric films, nor is it a thorough collection of different food packaging applications. It is also not a book that concentrates on physicochemical characterization methods and engineering aspects of packaging. Instead, this is a book that provides systematic knowledge about key methods of evaluating natural resources, agro-industrial wastes and by products for development of edible packaging, and concentrates on concepts, strategies, technologies, and applications of active edible packaging based solely on natural components. It is designed to share both positive and negative experiences in an emerging field that is expected to play a central role in improving food safety and quality, human health and environmentally friendly practices.
The physiological chirality of extracellular environments is substantially affected by pathological diseases. However, how this stereochemical variation drives host immunity remains poorly understood. Here, we report that pathology-mimetic M-nanofibrils—but not physiology-mimetic P-nanofibrils—act as a defense mechanism that helps to restore tissue homeostasis by manipulating immunological response. Quantitative multi-omics in vivo and in vitro showed that M-nanofibrils significantly inhibited inflammation and promoted tissue regeneration by upregulating M2 macrophage polarization and downstream immune signaling compared with P-nanofibrils. Molecular analysis and theoretical simulation demonstrated that M-chirality displayed higher stereo-affinity to cellular binding, which induced higher cellular contractile stress and activated mechanosensitive ion channel PIEZOl to conduct Ca²⁺ influx. In turn, the nuclear transfer of STAT was biased by Ca²⁺ influx to promote M2 polarization. These findings underscore the structural mechanisms of disease, providing design basis for immunotherapy with bionic functional materials.
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Tendon injuries are a global health problem that affects millions of people annually. The properties of tendons make their natural rehabilitation a very complex and long-lasting process. Thanks to the development of the fields of biomaterials, bioengineering and cell biology, a new discipline has emerged, tissue engineering. Within this discipline, diverse approaches have been proposed. The obtained results turn out to be promising, as increasingly more complex and natural tendon-like structures are obtained. In this review, the nature of the tendon and the conventional treatments that have been applied so far are underlined. Then, a comparison between the different tendon tissue engineering approaches that have been proposed to date is made, focusing on each of the elements necessary to obtain the structures that allow adequate regeneration of the tendon: growth factors, cells, scaffolds and techniques for scaffold development. The analysis of all these aspects allows understanding, in a global way, the effect that each element used in the regeneration of the tendon has and, thus, clarify the possible future approaches by making new combinations of materials, designs, cells and bioactive molecules to achieve a personalized regeneration of a functional tendon.
The tumor microenvironment (TME) has become the focus of interest in cancer research and treatment. It includes the extracellular matrix (ECM) and ECM-modifying enzymes that are secreted by cancer and neighboring cells. The ECM serves both to anchor the tumor cells embedded in it and as a means of communication between the various cellular and non-cellular components of the TME. The cells of the TME modify their surrounding cancer-characteristic ECM. This in turn provides feedback to them via cellular receptors, thereby regulating, together with cytokines and exosomes, differentiation processes as well as tumor progression and spread. Matrix remodeling is accomplished by altering the repertoire of ECM components and by biophysical changes in stiffness and tension caused by ECM-crosslinking and ECM-degrading enzymes, in particular matrix metalloproteinases (MMPs). These can degrade ECM barriers or, by partial proteolysis, release soluble ECM fragments called matrikines, which influence cells inside and outside the TME. This review examines the changes in the ECM of the TME and the interaction between cells and the ECM, with a particular focus on MMPs.
Le collagène est un élément majeur de l'architecture des organes chez les mammifères où il forme diverses structures tridimensionnelles (3D) propres à chaque tissu. La visualisation de cette organisation 3D multi-échelle est cruciale pour comprendre la structuration d'organes tels que la cornée ou la peau et guider l'ingénierie de tissus artificiels, qui doivent être structurés de manière appropriée pour être fonctionnels. L’organisation du collagène est de plus affectée dans de nombreuses pathologies. Caractériser ces désordres tissulaires in situ de manière quantitative constitue ainsi un enjeu biomédical majeur.La microscopie SHG est reconnue depuis plusieurs années comme la technique de référence pour imager le collagène fibrillaire in situ dans des tissus sans marquage, et ceci avec un excellent contraste. Cette thèse présente la mise en place et l'application de nouvelles modalités de microscopie SHG fondées sur la polarisation, visant à obtenir des paramètres fiables et quantitatifs pour décrire plus précisément la structure tridimensionnelle du collagène.Tout d’abord, nous présentons une modalité utilisant des polarisations incidentes linéaires (P-SHG) pour analyser l’organisation multi-échelle du collagène dans divers tissus, sains et pathologiques. Ces analyses ont été conduites sur des objets du patrimoine (parchemins, constitués de collagène de peaux animales) ainsi que sur des tissus biologiques (cornées). D’une part, tirant parti du caractère non invasif de cette modalité, nous caractérisons la dégradation du collagène dans des parchemins, précieux objets d’art et d’Histoire, démontrant ainsi l’intérêt de la microscopie SHG dans le domaine du patrimoine, notamment pour des diagnostics de l’état de conservation des objets riches en collagène. D’autre part, une imagerie quantitative de cornées humaines saines est présentée, et comparée à des cornées présentant un kératocône, pathologie courante aujourd’hui. Des modèles murins de kératocônes cornéens sont également étudiés, dans le but de valider leur pertinence.Enfin, une modalité utilisant des polarisations incidentes circulaires pour mesurer des signaux de dichroïsme circulaire (CD-SHG) est exposée. Dans un premier temps, nous présentons la mise en place expérimentale rigoureuse de cette modalité, en identifiant et corrigeant des artefacts typiques de cette technique. Dans un second temps, nous proposons une nouvelle approche théorique pour décrire les signaux de CD-SHG. Les simulations numériques de l’expression analytique obtenue sont comparées aux résultats expérimentaux, dans le but de comprendre l’évolution des signaux de CD-SHG en fonction de l’architecture 3D du collagène.
Collagen is an essential structural protein in animal tissues and plays key roles in cellular modulation. We investigated methods to discover collagen model peptides (CMPs) that would self-assemble into triple helices and then grow into supramolecular organizations with diverse morphological features, which would be valuable as biomaterials. This challenging undertaking was achieved by placing azobenzene groups on the ends of the CMPs, (GPO)n (n = 3–10), Azo-(GPO)n. In a dilute aqueous solution (80 μM), CD spectra indicated that the Azo-(GPO)n (n > 4) formed triple helices due to strong hydrophobic azobenzene interactions, and that helix stability was increased with the peptide segment length. The resulting triple helices induced a specific azobenzene orientation through turned and twisted configurations as shown by CD spectra. TEM observations for the same solutions disclosed the morphologies for the Azo-CMPs. Azo-(GPO)3, having the shortest peptide segment, showed no nanostructure, both Azo-(GPO)4 and Azo-(GPO)5 provided consistent well-developed nanofiber structures resembling the natural collagen fibers, and Azo-(GPO)ns (n = 6–10) grew into flexible rod-like micelle fibers. In addition, alkyl chain-attached CmAzo-(GPO)5 displayed a toroidal morphology, and Azp-deg-(GPO)5 having a hydrophilic spacer assembled into a bilayer vesicle structure. These diverse morphological features are considered to be due to the characteristics of the pre-organized triple helix units. Photo-isomerization of the azobenzene moiety brought about the disappearance of such characteristic nano-architectures. When the solution concentration was increased up to 1 wt%, only Azo-(GPO)4 and Azo-(GPO)5 spontaneously formed hydrogels exhibiting a satisfactory gel-to-sol transition upon UV irradiation.
Self-assembled peptides have been shown to form well-defined nanostructures which display outstanding characteristics for many biomedical applications and especially in controlled drug delivery. Such biomaterials are becoming increasingly popular due to routine, standardized methods of synthesis, high biocompatibility, biodegradability and ease of upscale. Moreover, one can modify the structure at the molecular level to form various nanostructures with a wide range of applications in the field of medicine. Through environmental modifications such as changes in pH and ionic strength and the introduction of enzymes or light it is possible to trigger self-assembly and design a host of different self-assembled nanostructures. The resulting nanostructures include nanotubes, nanofibers, hydrogels and nanovesicles which all display a diverse range of physico-chemical and mechanical properties. Depending on their design, peptide self-assembling nanostructures can be manufactured with improved biocompatibility and in-vivo stability and the ability to encapsulate drugs with the capacity for sustained drug delivery. These molecules can act as carriers for drug molecules to ferry cargo intracellularly and respond to stimuli changes for both hydrophilic and hydrophobic drugs. This review explores the types of self-assembling nanostructures, the effects of external stimuli on and the mechanisms behind the assembly process, and applications for such technology in drug delivery.
One key problem in understanding the biosynthesis of collagens remains the assembly of the three α-chains. How and when are the different gene products selected, aligned, and folded into a triple helix? As the spatial arrangement during biosynthesis might be important, we concentrated on whether the rough endoplasmic reticular membrane is involved in this process. Microsomes were prepared from biosynthetically labeled chick tendon fibroblasts. Vesicles were spread as a monomolecular film which was then transferred over several compartments of a filmbalance containing fresh subphase. Fluorograms of the surface film showed that the monolayer contains procollagen chains. When the monolayer was transferred onto a chymotrypsin/trypsin-containing subphase, the gel bands of the proα-chains were shifted into the position of mature α-chains, indicating that only the propeptides were digested and the collagenous regions were protected due to triple helix formation. Our results suggest that newly synthesized proα-chains can associate as trimers and fold into a triple helical conformation while they are still associated with the membranes of the rough endoplasmic reticulum. These processes also occur when interchain disulfide linkage is inhibited, indicating that chain selection and registration is not dependent on formation of covalent bonds among the carboxyl propeptides.
Portions of the amino acid sequences of four representative proteins containing alpha-helices arranged in a coiled-coil rope-like structure have been analysed in terms of the preference of the residues or residue types for specific positions within the observed heptad repeats. The results clearly show an asymmetric distribution of residues which can be interpreted in terms of the size and shape of the residue, the geometry of the coiled-coil structure, or the facility with which interchain or intermolecular interactions may be made. The statistical data reported here may also be used to predict regions of coiled-coil structure in other proteins.
We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.
A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.
Mammalian hibernation is a unique physiological adaptation that allows the sustainment of life under extremely low body temperatures. In the chipmunk, we found four proteins related specifically to hibernation. These proteins started to diminish in concentration in the blood before and disappeared during hibernation. These proteins reappeared in the blood as hibernation ceased and remained during nonhibernation. The complete or partial amino acid sequences of the four proteins showed that three (27-, 25-, and 20-kDa) were previously unknown, whereas another (55-kDa) is highly homologous with alpha 1-antitrypsin. The three novel proteins are homologous, indicating that they are a family. In the NH2-terminal regions of these proteins, a collagen-like amino acid sequence is present, whereas in their COOH-terminal regions, two sequences, Ser-Ala-Phe-Ala-Val-Lys and Val-Trp-Leu-Glu, are conserved. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and gel permeation chromatography under denaturating conditions revealed that the four proteins form a 140-kDa complex in the plasma fraction. The novel proteins were detected in blood of another hibernator, the ground squirrel, but not in rodent nonhibernators, namely tree squirrels and rats. The present finding is the first identification of a hibernation-specific protein. The presence of specific proteins in hibernators suggests the involvement of genetic factors in the control of hibernation. These proteins provide valuable tools for understanding molecular mechanisms of mammalian hibernation.
The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.
Pullulanase from Klebsiella pneumoniae strain FG9 has an unusual N-terminal amino acid sequence that includes six repeats of the tripeptide Gly-X-Pro. This type of sequence is characteristic of animal collagens and collagen-like proteins which form triple helical structures. We have investigated the molecular organization of this bacterial pullulanase isolated from the cell surface of Escherichia coli cells that carry the cloned FG9 pulA (pullulanase encoding) gene. Non-denaturing polyacrylamide gel analysis shows that pullulanase exists as higher order, apparently homogeneous, structures. We have used highly purified bacterial collagenase to probe the role of the collagen-like region and we demonstrate that this feature is essential for non-covalent association of pullulanase homotrimers. In addition we show collagenase-specific release of cell-bound pullulanase.
An early form of procollagen I was found in acetic acid extracts of radioactively labeled chick embryo skull bones. It resembled native procollagen I, but sedimented slightly faster, and its component chains were slightly underhydroxylated and were not disulfide-linked to each other, although its propeptides were internally disulfide-bonded. Pulse-chase experiments showed its conversion to disulfide-linked procollagen. As the same conversion occurred when proline hydroxylation was blocked by 2,2'-dipyridyl, we infer that the formation of this precursor from its component chains does not require collagen triple helix formation. We suggest that interaction between the folded carboxyl propeptides of individual pro-alpha (I) chains is an important step in the formation of this precursor and of procollagen I. Studies of the refolding and association of fully reduced and denatured carboxyl propeptides supported this concept. In the presence of glutathione the correct disulfide bonds could be reestablished, as judged by a mapping of some tryptic peptides. Individual carboxyl propeptides refolded first, and this occurred even in 2 M urea. Recognition between folded carboxyl propeptides occurred only when less than 0.5 M urea was present. The presence of the carboxyl telopeptides was important for trimeric reassembly. Individual propeptides also folded spontaneously during cell-free translation of pro-alpha (I) chains and were recognized by specific antibodies. We consider the role of carboxyl propeptides in the formation of procollagen I molecules and suggest a model of self-assembly, possibly facilitated by interactions with the luminal surface of the rough endoplasmic reticulum.
We have identified a fifth member of the alpha subfamily of vertebrate laminin chains. Sequence analysis revealed a close relationship of alpha 5 to the only known Drosophila alpha chain, suggesting that the ancestral alpha gene was more similar to alpha 5 than to alpha 1-4. Analysis of RNA expression showed that alpha 5 is widely expressed in adult tissues, with highest levels in lung, heart, and kidney. Our results suggest that alpha 5 may be a major laminin chain of adult basal laminae.
We have previously identified and purified transforming growth factor-beta 1 (TGF-beta 1)-binding proteins from porcine uterus membranes (Ichijo, H., Rönnstrand, L., Miyagawa, K., Ohashi, H., Heldin, C.-H., and Miyazono, K. (1991) J. Biol. Chem. 266, 22459-22464). One of these TGF-beta 1-binding proteins, with a molecular weight of 40,000, was purified to homogeneity and subjected to amino acid sequence analysis. The amino acid sequences obtained were used to isolate two closely related cDNA clones from a porcine uterus cDNA library. The deduced amino acid sequences revealed that both cDNAs encoded proteins that were mainly composed of fibrinogen-like and collagen-like domains. Therefore, they were denoted ficolin-alpha and ficolin-beta. Expression of ficolin-alpha and -beta cDNA in mammalian cells revealed that ficolin forms dimers, trimers, and several higher order of oligomers, whose molecular weights fit well with those of the purified TGF-beta 1-binding proteins from porcine uterus. Moreover, immunoblotting analysis using a peptide anti-serum against ficolin indicated that the TGF-beta 1-binding proteins identified in porcine uterus are ficolin-alpha, -beta, and their oligomers or closely related molecules. However, recombinant ficolin-alpha and -beta did not bind TGF-beta 1, despite the similarities in molecular weights and immunoreactivity with the material from the natural source. It is possible that a specific posttranslational modification of ficolin or interaction with another component is needed for TGF-beta 1 binding. Analysis by Northern blotting revealed that the expression of ficolin-alpha mRNA is relatively restricted and most abundant in placenta and lung. On the other hand, ficolin-beta was mainly expressed in skeletal muscle. The in vivo functions of ficolin will be discussed.
Fungal fimbriae are surface appendages that were first described on the haploid cells of the smut fungus, Microbotryum violaceum. They are long (1–20 microm), narrow (7 nm) flexuous structures that have been implicated in cellular functions such as mating and pathogenesis. Since the initial description, numerous fungi from all five phyla have been shown to produce fimbriae on their extracellular surfaces. The present study analyses the protein component of M.violaceum fimbriae. The N‐terminus and three internal amino acid sequences were determined. All four show a strong similarity to sequences which are characteristic of the collagen gene family. Enzymatic digests and immunochemical analyses support this finding. Based on these results, it is suggested that the proteinaceous subunits of fimbriae should be termed fungal collagens. Previously, collagen has been found only among members of the kingdom Animalia where it is the principal component of the animal extracellular matrix and is the most abundant animal protein. The unexpected finding of collagen in the members of the Mycota suggests that it may have evolved from a common ancestor that existed before the divergence of fungi and animals. Further, native fungal fimbriae can function as a mammalian extracellular matrix component. They can act as a substratum which permits animal cells to adhere, spread, and proliferate in a manner similar to animal collagens. The implications of this finding to both phylogeny and pathology are discussed.
The discontinuities found in heptad repeats of α-helical coiled-coil proteins have been characterized. A survey of 40 α-fibrous proteins reveals that only two classes of heptad breaks are prevalent: the stutter, corresponding to a deletion of three residues, and the newly identified “stammer,” corresponding to a deletion of four residues. This restriction on the variety of insertions/deletions encountered gives support to a unifying structural model, where different degrees of supercoiling accommodate the observed breaks. Stutters in the hemagglutinin coiled-coil region have previously been shown to produce an underwinding of the supercoil, and we show here how, in other cases, stammers would lead to overwinding. An analysis of main-chain structure also indicates that the mannose-binding protein, as well as hemagglutinin, contains an underwound coiled-coil region. In contrast to knobs-into-holes packing, these models give rise to non-close-packed cores at the sites of the heptad phase shifts. We suggest that such non-close-packed cores may function to terminate certain coiled-coil regions, and may also account for the flexibility observed in such long α-fibrous molecules as myosin. The local underwinding or overwinding caused by these specific breaks in the heptad repeat has a global effect on the structure and can modify both the assembly of the protein and its interaction properties. © 1996 Wiley-Liss, Inc.
This review summarizes the data on 278 different mutations found to date in the genes for types I, II, III, IX, X, and XI collagens from 317 apparently unrelated patients. A majority (217 mutations; 78% of the total) of the mutations are single-base and either change the codon of a critical amino acid (63%), or lead to abnormal RNA splicing (13%). Most of the amino acid substitutions are those of a bulkier amino acid for the obligatory glycine of the repeating-Gly-X-Y-sequence of the collagen triple helix (155; 56%). Altogether, 26 different mutations (9.4% of the mutations) occur in more than one unrelated individual. The 65 patients in whom the 26 mutations were characterized constitute almost one-fifth (20.5%) of the 317 patients analyzed. The mutations in types I, II, III, IX, X, and XI collagens cause a wide spectrum of diseases of bone, cartilage, and blood vessels, including osteogenesis imperfecta, a variety of chondrodysplasias, types IV and VII of the Ehlers-Danlos syndrome, and, rarely, some forms of osteoporosis, osteoarthritis, and familial aneurysms. Hum Mutat 9:300–315, 1997. © 1997 Wiley-Liss, Inc.
A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.
Lower vertebrates including cyclostomes, chondrichthyes and osteichthyes are usually referred to as fish. They are Poikilothermic and are to be found in the wider range of habitats from below zero to over 50°C, The collagenous proteins of fish, as well as mammals, are widely distributed in skin, cartilage, bone and many other organs. About 30 years ago, studies of fish had contributed substantially to our understanding of the macromolecular structure of collagen, because their connective tissues have some unique properties. First, the skin and swim bladder collagens of fish are extremely soluble in dilute acid to yield a large amount of soluble protein; this provides a good source for the structural study of collagen molecules in solution. Perhaps the carp swim bladder collagen, which was called “ichthyocol”, was the first soluble collagen to be fully characterized with respect to its molecular weight and molecular dimensions in native and denatured states (Boedtker and Doty, 1956). At present, the basic macro-molecular structure of interstitial collagens is considered to be common to all vertebrate species.
Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen
in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3′ end of the EDA gene. All transcripts recovered have the same 5′ exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A
may correlate with differential roles during embryonic development.
An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless,
most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase
activity. Pro-α1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to
contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the T of the triple helices was only about 32-34°C. Coexpression of the pro-α1(III) chains with the α and β subunits of human prolyl
4-hydroxylase increased the T to about 40°C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was
also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-α1(III) chains and α1(III) chains were
found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect
cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced
up to about 80 μg of cellular type III collagen (120 μg of procollagen) per 5 × 106 cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The
4-hydroxyproline content and T of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine
content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.
In this communication, we report the use of a conformationally constrained organic template to induce collagen-like triple helical structures composed of Gly-Pro-Hyp sequences. This template, cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid (known as the Kemp triacid, KTA), possesses three carboxyl groups which can be coupled to the N-terminal of three peptide chains. We insert as a spacer between each peptide chain and each carboxyl group on KTA to compensate for the difference in diameters between the KTA and the collagen triple helix and to facilitate the synthesis. On the basis of the NMR and optical rotation results we can conclude that KTAg-3,3 forms a triple-helical conformation in H2O with a melting temperature of 30°C. We believe this compound represents the shortest chain polypeptide able to form a triple helical structure at room temperature in H2O reported to date. This compound will be useful as a model system for the study of the stages of triple-helix folding. Therefore, we denote this borderline ordered structure as an incipient triple helix.
This review summarizes the data on 278 different mutations found to date in the genes for types I, II, III, IX, X, and XI collagens from 317 apparently unrelated patients. A majority (217 mutations; 78% of the total) of the mutations are single-base and either change the codon of a critical amino acid (63%), or lead to abnormal RNA splicing (13%). Most of the amino acid substitutions are those of a bulkier amino acid for the obligatory glycine of the repeating-Gly-X-Y-sequence of the collagen triple helix (155; 56%). Altogether, 26 different mutations (9.4% of the mutations) occur in more than one unrelated individual. The 65 patients in whom the 26 mutations were characterized constitute almost one-fifth (20.5%) of the 317 patients analyzed. The mutations in types I, II, III, IX, X, and XI collagens cause a wide spectrum of diseases of bone, cartilage, and blood vessels, including osteogenesis imperfecta, a variety of chondrodysplasias, types IV and VII of the Ehlers-Danlos syndrome, and, rarely, some forms of osteoporosis, osteoarthritis, and familial aneurysms. Hum Mutat 9:300–315, 1997. © 1997 Wiley-Liss, Inc.
The collectins are a group of mammalian lectins containing collagen-like regions. They include mannan binding protein, bovine conglutinin, lung surfactant protein A, lung surfactant protein D, and a newly discovered bovine protein named collectin-43. These proteins share a very similar modular domain composition and overall 3-dimensional structure. They also appear to play similar biological roles in the preimmune defense against microorganisms in both serum and lung surfactant. The close evolutionary relationship between the collectins is further emphasized by a common pattern of exons in their genomic structures and the presence of a gene cluster on chromosome 10 in humans that contains the genes known for the human collectins. Studies on the structure/function relationships within the collectins could provide insight into the properties of a growing number of proteins also containing collagenous regions such as Clq, the hibernation protein, the α- and β-ficolins, as well as the membrane acetylcholinesterase and the macrophage scavenger receptor.
This paper describes in detail our work on the structure of collagen which we have already outlined elsewhere (Rich & Crick, 1955). The main substance of the paper is:(1)a demonstration that, given certain assumptions, only two basic types of structures are possible for collagen;(2)detailed work on the coordinates and Fourier transforms of one of these models (collagen II), and a comparison between these predictions and the observed X-ray diffraction data.
Folding of the collagen triple helix provides an opportunity to look at multichain molecular assembly. This triple helix also offers unique advantages for the study of folding because the process is very slow compared to globular proteins, and the kinetics of folding can be obtained in real time by NMR. Studies on triple-helical peptides illustrate the ability to observe kinetic folding intermediates directly and the ability to propose detailed mechanisms of folding through the use of real-time NMR methods. Defective collagen folding has been implicated in various connective tissue diseases and the capacity of NMR to look at the folding of specific sites provides a tool for obtaining information about altered folding mechanisms. Comparison of folding in peptides that model normal and diseased collagens could shed light on the molecular perturbation and the etiology of disease.
An improved α-diagram has been obtained from a native molluscan “catch” muscle (the anterior byssal retractor muscle of Mytilus edulis). The most striking aspect of the diagram is a strong near-equatorial layer line at about 89 Å. The 5·1 Å spacing is meridional. These features are accounted for by a coiled-coil α-helical structure. Computations indicate that the best fit is obtained with a two-chain structure when various types of disorder are assumed. A possible model is described having a densely packed hydrophobic region between the chains, and most of the polar groups pointing out into the solvent. An implication of the coiled-coil structure is that the α-helix in proteins requires stabilization by side-chain interactions.
Quantitative X-ray diffraction data have been collected from stretched kangaroo tail tendon and used to test models for the conformation of the polypeptide chains in the collagen molecule. The magnitude of the unit twist of the molecular helix was estimated to be 107.1 ° ± 0.6 °, which is close to the value expected for a helix with ten units in three turns. The intensity data were used to carry out a linked-atom least-squares refinement of models based on two possible interchain hydrogen bonding schemes suggested by Rich & Crick (1955, 1961). No stereochemically acceptable solution could be found for the hydrogen bonding scheme of model I, but a stereochemically satisfactory solution was found for the scheme of model II which gave a crystallographic R factor of 0.272.
The collagen-like peptides (L-Pro-L-Pro-Gly)n and (L-Pro-L-Hyp-Gly)n with n = 5 and 10, were examined in terms of their triple helix ⇌ coil transitions in aqueous and nonaqueous solvents. The peptides were soluble in 1,2-propanediol containing 3% acetic acid and they were found to form triple-helical structures in this solvent system. The water content of the solvent system and the amount of water bound to the peptides were assayed by equilibrating the solvent with molecular sieves and carrying out Karl Fischer titrations on the solvent phase. After the solvent was dehydrated, much less than one molecule of water per tripeptide unit was bound to the peptides. Since the peptides remained in a triple-helical conformation, the results indicated that water was not an essential component of the triple-helical structure. Comparison of peptides with the same chain length demonstrated that the presence of hydroxyproline increased the thermal stability of the triple helix even under anhydrous conditions. The results, therefore, did not support recent hypotheses that hydroxyproline stabilizes the triple helix of collagen and collagen-like peptides by a specific interaction with water molecules. Analysis of the thermal transition curves in several solvent systems showed that although the peptides containing hydroxyproline had tm values which were 18.6° to 32.7°C higher, the effect of hydroxyproline on ΔG was only 0.1 to 0.3 kcal per tripeptide unit at 25°C. The results suggested, therefore, that the influence of hydroxyproline on helical stability may be explained by intrinsic effects such as dipole–dipole interactions or by changes in the solvation of the peptides by alcohol, acetic acid, and water. A direct calorimetric measurement of the transition enthalpy for (L-Pro-L-Pro-Gly)n in 3% or 10% acetic acid gave a value of −1.84 kcal per tripeptide unit for the coil-to-helix transition. From the value for enthalpy and from data on the effects of different chain lengths on the thermal transition, it was calculated that the apparent free energy for nucleation was +5 kcal/mol at 25°C (apparent nucleation parameter = 2 × 10−4M−2). The value was dependent on solvent and on chemical modification of end groups.
We have used molecular replacement followed by a highly parameterized refinement to determine the structure of tropomyosin crystals to a resolution to 9 A. The shape, coiled-coil structure and interactions of the molecules in the crystals have been determined. These crystals have C2 symmetry with a = 259.7 A, b = 55.3 A, c = 135.6 A and beta = 97.2 degrees. Because of the unusual distribution of intensity in X-ray diffraction patterns from these crystals, it was possible to solve the rotation problem by inspection of qualitative aspects of the diffraction data and to define unequivocally the general alignment of the molecules along the (332) and (3-32) directions of the unit cell. The translation function was then solved by a direct search procedure, while electron microscopy of a related crystal form indicated the probable location of molecular ends in the asymmetric unit, as well as the anti-parallel arrangement. The structural model we have obtained is much clearer than that obtained previously with crystals of extraordinarily high solvent content and shows the two alpha-helices of the coiled coil over most of the length of the molecules and establishes the coiled-coil pitch at 140(+/- 10) A. Moreover, the precise value of the coiled-coil pitch varies along the molecule, probably in response to local variations in the amino acid sequence, which we have determined by sequencing the appropriate cDNA. The crystals are constructed from layers of tropomyosin filaments. There are two molecules in the crystallographic asymmetric unit and the molecules within a layer are bent into an approximately sinusoidal profile. Molecules in consecutive layers in the crystal lie at an angle relative to one another as found in crystalline arrays of actin and myosin rod. There are three classes of interactions between tropomyosin molecules in the spermine-induced crystals and these give some insights into the molecular interactions between coiled-coil molecules that may have implications for assemblies such as muscle thick filaments and intermediate filaments. In interactions within a layer, the geometry of coiled-coil contacts is retained, whereas in contacts between molecules in adjacent layers the coiled-coil geometry varies and these interactions instead appear to be dominated by the repeating pattern of charged zones along the molecule.
Members of the spectrin superfamily of proteins contain different numbers of homologous repeats arranged in tandem. Each of these consists of a three-alpha-helix motif, comprising two similarly and one oppositely directed alpha-helical segment joined by nonhelical linkers of characteristic length. The right-handed alpha-helices each display a heptad repeat in their amino acid sequences indicative of left-handed coiled-coil-like packing. We have calculated the potential number of inter-helix ionic interactions that specify the spatial arrangement of the helices in the motif in terms of both the handedness of helix connectivity (left or right) and the relative axial stagger between the three alpha-helices. All of the models examined were constrained to have optimal coiled-coil packing. For alpha-spectrin and alpha-actinin the results provide strong support for a left-handed connectivity of the three helices and axial repeat lengths of 5.05 and 6.24 nm, respectively. Furthermore, the axial staggers between homologous segments in the preferred models are identical. The insights provided into the topography of this widespread tertiary fold may prove of value to those concerned with the problem of de novo protein design.
Bacteriophage PRD1 infecting Escherichia coli and Salmonella typhimurium translocates its membrane from the host plasma membrane to the virus particle. One obligatory component in this process is the major capsid protein. In this investigation we describe characteristics of the homomultimeric major and minor capsid proteins including the sequences of the corresponding genes. The minor capsid protein was found to contain a short collagen-like region (Gly-X-Y)6. This is the first time this motif has been reported for a prokaryotic protein.
The cell adhesion, spreading and neurite-promoting properties of mouse tumor laminin fragment E8, which contains major site(s) responsible for laminin-cell interactions, were probed by proteolytic degradation, denaturation, synthetic peptides and antibody inhibition. Removal of more than half of the N-terminal portion contributing to the rod-like domain did not effect cell attachment or spreading although neurite-promoting activity was reduced. More extensive degradation of the rod or of the globular domains of E8, or separation of the globule from the rod, also resulted in loss of cell spreading activity although weak attachment was found to an A chain subfragment comprising the globular domain and a short piece of the rod. Exposure of E8 to increasing concentrations of dissociating agents produce an apparently reversible denaturation but an irreversible loss of both attachment and neurite-promoting activities, as did reduction and alkylation of disulfide bonds in the globular domain. Although cell adhesion and spreading were blocked by antibodies to an alpha 6 integrin subunit, neurite outgrowth was unaffected, indicating two distinct receptors for these two activities. Furthermore, a synthetic peptide, the sequence of which is found in the vicinity of adhesion and neurite-promoting sites and previously implicated in neurite growth and cell attachment activities, was found to be inactive. These results indicate that the major cell attachment and neurite-promoting sites of laminin are distinct although both require the native conformation of parts of the rod and the terminal globular domain of the long arm of laminin.
The macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an alpha-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.
The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the alpha-helical regions of the A, B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual alpha-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, alpha-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains. Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather than it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the alpha-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.
A tentative amino-acid sequence for the COOH-terminal half of rabbit skeletal tropomyosin is reported. These studies confirm our previous conclusions that this tropomyosin consists of several different but similar polypeptide chains. In the sequence, nonpolar residues occur in two series at intervals of seven residues. Amino-acid residues in series I are three residues on the NH(2)-terminal side of, and four residues on the COOH-terminal side of, residues in series II. The presence of occasional charged or ambivalent residues in the positions of series I or II does not lead to a disruption of this long-range pattern. The majority of residues located between the nonpolar residues are charged or polar amino acids. Two highly similar or identical alpha-helices with the reported sequence can be packed together in parallel in a coiled-coil structure. These may be in register or staggered by seven residues or some multiple of it. The observation that groups of small hydrophobic side chains appear to alternate with groups of bulky side chains suggests that a staggered arrangement of the two alpha-helices would maximize the regularity and hydrophobic interactions of the coiled-coil. Model building considerations show that this would occur with a stagger of 14 residues. Such an arrangement could account for the end-to-end aggregation of tropomyosin in solution, and in crystal and tactoid filaments. However, a structure in which the two polypeptides are in register cannot be ruled out.
The effect of the disulfide cross-linkages on the stability and regeneration of the collagen-fold conformation in polypeptide chains of Ascaris collagen has been investigated. Cleavage of the disulfide cross-links lowers the helix-coil thermal transition temperature, Tm, about 19° and reduces the helix regeneration rate by a factor of 10-20 below that of the native collagen. Reoxidation following reductive cleavage of the SS bridges increases the regeneration rate to a level comparable to the native collagen and elevates Tm to an intermediate value between the native and fully reduced systems. The mechanism of formation and stabilization of the collagen fold in the reduced carboxymethylated polypeptide chains of Ascaris collagen (RCM Ascaris) has been examined. The rate of renaturation is shown to be independent of concen-tration over about a 500-fold range. Kinetic analysis of tritium-hydrogen-exchange curves reveals that approximately 230 slowly exchanging hydrogens/subunit chain (mol wt 62,000) are formed during the regeneration of the polyproline II type helix, in good agreement with the number determined for the native collagen per helical triplet. Tryptic digestion experiments indicate that a large portion (75-85%) of the renaturated RCM Ascaris chain remains intact at 5° following proteolysis. These results, when taken in conjunction with the invariance in molecular weight observed during transformation of the helix to the coil forms, suggest that the subunit of Ascaris is a single polypeptide chain which folds back upon itself to form a stable, collagen-type triple helix at low temperature.
(Pro-Pro-Gly)10 forms single crystals, providing X-ray diffraction data to 0.22 nm resolution. In the crystals, the polypeptides form triplexes that aggregate end-to-end in quasi-infinite helices with axial translation per tripeptide h = 0.287 nm and the corresponding rotation t = −102.9 °. The structure, which may be an allomorph of collagen, has been refined by the linked-atom least-squares procedure. In addition, three water molecules per tripeptide have been detected by Fourier difference syntheses. One of them forms an intrachain hydrogen-bonded bridge O(Pro2) - - - W - - - O(Gly). There are also interchain hydrogen bonds (Gly)NH - - - O(Pro1) within the triplex.
Human mannose-binding protein is a hexamer of trimers with each subunit consisting of an amino-terminal region rich in cysteine, 19 collagen repeats, a 'neck', and a carbohydrate recognition domain that requires calcium to bind ligand. A 148-residue peptide, consisting of the 'neck' and carbohydrate recognition domains forms trimers in solution and in crystals. The structure of this trimeric peptide has been determined in two different crystal forms. The 'neck' forms a triple alpha-helical coiled-coil. Each alpha-helix interacts with a neighbouring carbohydrate recognition domain. The spatial arrangement of the carbohydrate recognition domains suggest how MBP trimers form the basic recognition unit for branched oligosaccharides on microorganisms.