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Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25
Abstract
Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B1-25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B1-25 was compared with that of a truncated homodimer (dSP-B8-25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at ∼ 500 cm− 1 indicated a stable, although highly strained disulfide conformation with a χ(CS-SC) dihedral angle of ± 10° for the dSP-B1-25 dimer. In contrast, the truncated dimer dSP-B8-25 exhibited a series of disulfide conformations with the χ(CS-SC) dihedral angle taking on values of either ± 30° or 85± 20°. For conformations with χ(CS-SC) close to the ± 90° value, the Raman spectra of the 8-25 truncated dimers exhibited χ(SS-CC) dihedral angles of 90/180° and 20-30°. In the presence of a lipid mixture, both constructs showed a ν(S-S) band at ∼ 488 cm− 1, corresponding to a χ(CS-SC) dihedral angle of ± 10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼ 60-70° to the surface normal, while the β structure had ∼ 40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.
... (dSP-B1-25) with Raman spectroscopy. 224 This protein is a homodimer with three intramolecular disulfide bonds and one intermolecular disulfide bridge. ...
... Furthermore, the Raman spectrum of the capsules shows an additional peak at 709 cm -1 , which refers to the C-S stretching vibration of the N-C-C-S-S moieties with N and S-S in the trans-conformation. 224 This is another hint for the intermolecular cross-linking in the shells causing the conformational changes in the BSA molecules after the capsule formation. ...
... In another approach, the Raman shift of the S-S stretching vibration is dependent on the dihedral angle between the planes containing the Cβ-S and the S-Cβ´ bonds of the Cα-Cβ-S-S-Cβ´-Cα´ group, respectively. 221-223 Disulfide bonds with an S-S stretching vibration with a value near 510 cm -1 should have a Cβ-S-S-Cβ´ dihedral angle of ±90°.224 Although the mostly observed S-S stretching vibrations exist between 500 and 540 cm -1 , the strained disulfide groups with dihedral angles less than ±65° are also possible. ...
Diese Doktorarbeit behandelt die Synthese von Protein- und kompositen Protein-Mineral-Mikrokapseln durch die Anwendung von hochintensivem Ultraschall an der Öl-Wasser-Grenzfläche. Während ein System durch BSA-Moleküle stabilisiert wird, wird das andere System durch verschiedene mit BSA modifizierten Nanopartikeln stabilisiert. Sowohl von allen Synthesestufen als auch von den resultierenden Kapseln wurden umfassende Untersuchungen durchgeführt und eine plausible Erklärung für den Mechanismus der Kapselbildung wurde vorgestellt. Während der Bildung der BSA-Mikrokapseln adsorbieren die Proteinmoleküle als Erstes an der O/W-Grenzfläche, entfalten sich dort und bilden ein Netzwerk, das durch hydrophobe Wechselwirkungen und Wasserstoffbrückenbindungen zwischen den benachbarten Molekülen stabilisiert wird. Gleichzeitig bewirkt die Ultraschallbehandlung die Quervernetzung der BSA-Moleküle über die Bildung von intermolekularen Disulfidbindungen. In dieser Doktorarbeit werden die experimentellen Nachweise für die durch Ultraschall induzierte Quervernetzung von BSA in den Schalen der proteinbasierten Mikrokapseln aufgezeigt. Deshalb wurde das Konzept, das vor vielen Jahren von Suslick und seinen Mitarbeitern vorgestellt wurde, zum ersten Mal durch experimentelle Nachweise bestätigt. Außerdem wurde ein konsistenter Mechanismus für die Bildung der intermolekularen Disulfidbindungen in der Kapselschale vorgestellt, der auf der Neuverteilung der Thiol- und Disulfidgruppen in BSA unter der Wirkung von hochenergetischem Ultraschall basiert. Auch die Bildung von kompositen Protein-Mineral-Mikrokapseln, die mit drei verschiedenen Ölen gefüllt wurden und deren Schalen aus Nanopartikeln bestehen, war erfolgreich. Die Beschaffenheit des Öls und die Art der Nanopartikel in der Schale hatten Einfluss auf die Größe und Form der Mikrokapseln. Die Untersuchung der kompositen Kapseln zeigte, dass die BSA-Moleküle, die an der Oberfläche der Nanopartikel in der Kapselschale adsorbiert sind, nicht durch intermolekulare Disulfidbindungen quervernetzt sind. Stattdessen findet die Bildung einer Pickering-Emulsion statt. Die Oberflächenmodifizierung der kompositen Mikrokapseln durch Vormodifizierung der Hauptbestandteile und auch durch Postmodifizierung der Oberfläche der fertigen kompositen Mikrokapseln wurde erfolgreich demonstriert. Zusätzlich wurden die mechanischen Eigenschaften beider Kapselarten verglichen. Dabei erwiesen sich die Protein-Mikrokapseln widerstandsfähiger gegenüber elastischer Deformation.
... [44][45][46] Disulde bonds with an S-S stretching vibration with a value near 510 cm À1 should have a Cb-S-S-Cb 0 dihedral angle of AE90 . 47 Although the mostly observed S-S stretching vibrations exist between 500 and 540 cm À1 , the strained disulde groups with dihedral angles less than AE65 are also possible. ...
... Biswas and coworkers observed a peak at 486 cm À1 while investigating the surfactant protein B (dSP-B 1-25 ) with Raman spectroscopy. 47 This protein is a homodimer with three intramolecular disulde bonds and one intermolecular disulde bridge. On the basis of several interpretations proposed by these authors for the spectrum of protein B, we suggest the following explanation for our system: the peak position at 487 cm À1 indicates that there is a highly strained but stable disulde bond conformation with a dihedral angle in the range of AE10 due to the crosslinking of the neighboring protein molecules in the capsule shell. ...
... Furthermore, the Raman spectrum of the capsules shows an additional peak at 709 cm À1 , which refers to the C-S stretching vibration of the N-C-C-S-S moieties with N and S-S in the trans-conformation. 47 This is another hint for the intermolecular cross-linking in the shells causing the conformational changes in the BSA molecules aer the capsule formation. Additionally, the capsules spectrum shows a band around 1450 cm À1 , which is assigned to the CH 2 and CH 3 bending vibration, and one around 1410 cm À1 corresponding to the C]O stretching vibration of dissociated carboxyl groups. ...
In this paper, we propose a consistent mechanism of protein microcapsule formation upon ultrasound treatment. Aqueous suspensions of bovine serum albumin (BSA) microcapsules filled with toluene are prepared by use of high-intensity ultrasound following a reported method. Stabilization of the oil-in-water emulsion by the adsorption of the protein molecules at the interface of the emulsion droplets is accompanied by the creation of the cross-linked capsule shell due to formation of intermolecular disulfide bonds caused by highly reactive species like superoxide radicals generated sonochemically. The evidence for this mechanism, which until now remained elusive and was not proven properly, is presented based on experimental data from SDS-PAGE, Raman spectroscopy and dynamic light scattering.
... In the Raman spectra and its colour maps, three peaks at 488, 958 and 2934 cm −1 were identified, which corresponded to those of proteins and lipids [30][31][32][33] . In addition, a number of small peaks were detected in the area of 1000-1600 cm −1 (Fig. 5b, c). ...
... In order to determine the elements of the PM2.5 used here, we employed a SEM-EDX system and found that the PM2.5 was in fact primarily consisted of carbon, sulfur, phosphorus, aluminium, calcium and magnesium (Fig. 6e, f). Carbon of the PM2.5 shows strong Raman peaks at 1354 and 1575 cm -1 (Supplementary Figure 3) 29 , different from carbon atoms in cell components such as proteins, fat and DNA [30][31][32][33] . Therefore, the carbon peaks above are highly likely due to the PM2.5 inside the cells. ...
PM2.5 has been correlated with risk factors for various diseases and infections. It promotes tissue injury by direct effects of particle components. However, effects of PM2.5 on cells have not been fully investigated. Recently, we developed a novel imaging technology, scanning electron-assisted dielectric-impedance microscopy (SE-ADM), which enables observation of various biological specimens in aqueous solution. In this study, we successfully observed PM2.5 incorporated into living mammalian cells in culture media. Our system directly revealed the process of PM2.5 aggregation in the cells at a nanometre resolution. Further, we found that the PM2.5 aggregates in the intact cells were surrounded by intracellular membrane-like structures of low-density in the SE-ADM images. Moreover, the PM2.5 aggregates were shown by confocal Raman microscopy to be located inside the cells rather than on the cell surface. We expect our method to be applicable to the observation of various nanoparticles inside cells in culture media.
... While there are no high-resolution crystal or NMR structures of the native SP-B protein, the structure of the NH 2 terminus up to residue 25 is well defined as determined by a combination of NMR, FTIR, and Raman spectroscopy (6,22,38,53,59,71). Residues 1-9 (FPIPLPYCW), proposed to act as an "insertion sequence," comprise a hydrophobic region containing a poly-proline-like sequence. ...
... This proposed splayed structure of the helix dimer is supported by Raman and polarized FTIR spectroscopy studies performed on the SP-B 1-25 and SP-B 8 -25 disulfide linked dimer. While the disulfide linkage is strained, it can clearly permit dimer helical splays and surface dependent helix orientation in surfactant lipids, in agreement with our proposed model (6). Additionally, recent work on lung surfactant peptoids, or peptide mimics, showed that the linkage of monomers by a rigid triazole moiety to form a dimer enhanced in vitro surface activity in a lipid film relative to its monomeric counterparts (16). ...
Lung surfactant protein B (SP-B) is required for proper surface activity of pulmonary surfactant. In model lung surfactant lipid systems composed of saturated and unsaturated lipids, the unsaturated lipids are removed from the film at high compression. It is thought that SP-B helps anchor these lipids closely to the monolayer in three-dimensional cylindrical structures termed "nanosilos" seen by atomic force microscopy imaging of deposited monolayers at high surface pressures. Here we explore the role of the SP-B NH(2) terminus in the formation and stability of these cylindrical structures, specifically the distribution of lipid stack height, width, and density with four SP-B truncation peptides: SP-B 1-25, SP-B 9-25, SP-B 11-25, and SP-B 1-25Nflex (prolines 2 and 4 substituted with alanine). The first nine amino acids, termed the insertion sequence and the interface seeking tryptophan residue 9, are shown to stabilize the formation of nanosilos while an increase in the insertion sequence flexibility (SP-B 1-25Nflex) may improve peptide functionality. This provides a functional understanding of the insertion sequence beyond anchoring the protein to the two-dimensional membrane lining the lung, as it also stabilizes formation of nanosilos, creating reversible repositories for fluid lipids at high compression. In lavaged, surfactant-deficient rats, instillation of a mixture of SP-B 1-25 (as a monomer or dimer) and synthetic lung lavage lipids quickly improved oxygenation and dynamic compliance, whereas SP-B 11-25 surfactants showed oxygenation and dynamic compliance values similar to that of lipids alone, demonstrating a positive correlation between formation of stable, but reversible, nanosilos and in vivo efficacy.
... Regarding the S-S bond of PEDPA, upon the FTIR measurements and comparing with data from the literature [76,77], we conclude that the corresponding FTIR is located in the range of wavenumbers around 500 cm − 1 and, also, below that (Fig. 4a). The peak can be resolved also for PEDPA contents ≥20 wt% in the blends. ...
A series of polymer blends based on poly(lactic acid), PLA, and poly(3,3-ethylene dithiodipropionate) PEDPA, varying in the PLA/PEDPA ratio from 90/10 to 50/50, are studied in this work. The said systems are envisaged for transdermal drug carrier systems, moreover, to be prepared using the method of electrospinning and exhibiting self-healing capabilities. The study involves calorimetry, for glass transition and crystallization, and dielectric spectroscopy, for molecular dynamics, as the main investigation tools, supplemented by X-ray diffraction, polarized light and Fourier transform infrared spectroscopies. For comparison, we study neat PLA and PEDPA. Actually, for PEDPA, the local and segmental molecular mobility map is shown here for the first time. The two phases were found to affect each other’s mobility, at a moderate extent. We conclude to the partial miscibility of the two polymers, resulting in systems with PLA matrices and PEDPA/PLA entities (drops) well dispersed throughout the matrix. The segmental dynamics of PLA accelerates in the blends, while the chain fragility (cooperativity) is suppressed, the effects indicating together a plasticization effect. On the other hand, the opposite results are recorded for the mobility of PEDPA at the presence of PLA, namely, deceleration and slight increase in fragility. Based on the ionic conductivity recordings, the PEDPA phase does not seem continuous throughout the blends’ volume at any case of PEDPA fraction. In general, the crystallization of PLA is suppressed in the blends, in both the nucleation and crystalline fraction, and, subsequently, the melting point drops. Serious changes in the semicrystalline morphology are observed within the blends, in terms of smaller spherulites and of smaller density. Overall, the results suggest that the PLA/PEDPA blends offer potentials for tuning the macroscopic materials’ performance, while the said combination of experimental techniques is able to provide useful information and indirect evidence regarding the polymers’ topology.
... Although hydrogel constructs were obtained using widely known chemical reactions with fully revealed mechanisms, further chemical analysis of different hydrogels with FTIR was conducted. The collagen hydrogel, PEG (NHS) hydrogel, PEG (SMPT) hydrogel, and hybrid hydrogels (PEG (NHS)-Collagen and PEG (SMPT)-Collagen) were distinguished from each other by their characteristic bands [35][36][37][38][39], while the two hybrid hydrogels had analogous IR spectra ( Figure S1). ...
Hydrogels are widely used in stem cell therapy due to their extensive tunability and resemblance to the extracellular matrix (ECM), which has a three-dimensional (3D) structure. These features enable various applications that enhance stem cell maintenance and function. However, fast and simple hydrogel fabrication methods are desirable for stem cells for efficient encapsulation and to reduce adverse effects on the cells. In this study, we present a one-pot double-crosslinked hydrogel consisting of polyethylene glycol (PEG) and collagen, which can be prepared without the multi-step sequential synthesis of each network, by using bio-orthogonal chemistry. To enhance the adipogenic differentiation efficiency of adipose-derived stem cells (ADSCs), we added degradable components within the hydrogel to regulate matrix stiffness through cell-mediated degradation. Bio-orthogonal reactions used for hydrogel gelation allow rapid gel formation for efficient cell encapsulation without toxic by-products. Furthermore, the hybrid network of synthetic (PEG) and natural (collagen) components demonstrated adequate mechanical strength and higher cell adhesiveness. Therefore, ADSCs grown within this hybrid hydrogel proliferated and functioned better than those grown in the single-crosslinked hydrogel. The degradable elements further improved adipogenesis in ADSCs with dynamic changes in modulus during culture and enabled the retrieval of differentiated cells for potential future applications.
... The 485 cm −1 band is attributed to the sulfur-containing proteins (cysteine/disulfide-rich; CREMPs) of the membranes ( Figure 6, Table 4). Table 4. Table 4. Raman vibrational bands observed in the ESM (cm −1 ; m medium, s strong, v very, w weak); table based on the assignments in [116][117][118][119][120][121][122][123]. The diffractogram of ESM, manually removed from the shell and air-dried on a flat support, is present in Figure 7. ...
The physicochemical features of the avian eggshell membrane play an essential role in the process of calcium carbonate deposition during shell mineralization, giving rise to a porous mineralized tissue with remarkable mechanical properties and biological functions. The membrane could be useful by itself or as a bi-dimensional scaffold to build future bone-regenerative materials. This review focuses on the biological, physical, and mechanical properties of the eggshell membrane that could be useful for that purpose. Due to its low cost and wide availability as a waste byproduct of the egg processing industry, repurposing the eggshell membrane for bone bio-material manufacturing fulfills the principles of a circular economy. In addition, eggshell membrane particles have has the potential to be used as bio-ink for 3D printing of tailored implantable scaffolds. Herein, a literature review was conducted to ascertain the degree to which the properties of the eggshell membrane satisfy the requirements for the development of bone scaffolds. In principle, it is biocompatible and non-cytotoxic, and induces proliferation and differentiation of different cell types. Moreover, when implanted in animal models, it elicits a mild inflammatory response and displays characteristics of stability and biodegradability. Furthermore, the eggshell membrane possesses a mechanical viscoelastic behavior comparable to other collagen-based systems. Overall, the biological, physical, and mechanical features of the eggshell membrane, which can be further tuned and improved, make this natural polymer suitable as a basic component for developing new bone graft materials.
... In TYR KO cells, there were no melanosomes, so we increased the laser intensity to 2 mW. When Raman measurements were performed with a laser intensity of 2 mW, a lipid peak of 2909 cm -1 [26,27] was observed without carbon peak (Fig. 3O). Therefore, we proved that the very dark particles in MNT-1 cells correspond to melanosomes containing melanin. ...
Melanins are the main pigments found in mammals. Their synthesis and transfer to keratinocytes have been widely investigated for many years. However, analysis has been mainly carried out using fixed rather than live cells. In this study, we have analysed the melanosomes in living mammalian cells using newly developed scanning electron-assisted dielectric microscopy (SE-ADM). The melanosomes in human melanoma MNT-1 cells were observed as clear black particles in SE-ADM. The main structure of melanosomes was toroidal while that of normal melanocytes was ellipsoidal. In tyrosinase knockout MNT-1 cells, not only the black particles in the SE-ADM images but also the Raman shift of melanin peaks completely disappeared suggesting that the black particles were really melanosomes. We developed a deep neural network (DNN) system to automatically detect melanosomes in cells and analysed their diameter and roundness. In terms of melanosome morphology, the diameter of melanosomes in melanoma cells did not change while that in normal melanocytes increased during culture. The established DNN analysis system with SE-ADM can be used for other particles, e.g. exosomes, lysosomes, and other biological particles.
... When the dihedral angle of ACACASASA segment in molecular structure was ±90°or 180°, the characteristic peaks of disulfide bonds will be located near 510 cm À1 . If the dihedral angle changed significantly, the peak will move to the range of 525-540 cm À1 [42][43][44]. ...
The R&D and application of natural biomass resources is one of the important ways to solve the world's energy shortage and environmental pollution. However, the complex processing technology and poor mechanical properties seriously hinder the development of the biomass polymers. A kind of natural antioxidant, alpha-lipoic acid, was used to modify wool keratin by grafting thiols in this study, so as to ameliorated the physical properties of regenerated materials. The optimal process parameters and modification mechanism were explored. Research results indicated that grafting ratio of lipoic acid can reach more than 13% and the polypeptide chains were more stretched in solution, when 30% lipoic acid was added into keratin solution at 40℃ with a reaction time of 2h. In addition, lipoic acid grafted on thiol groups can effectively weaken the intra- and intermolecular forces of keratin, this change made the arrangement of macromolecular chains more orderly. The physical structure of modified keratin became uniform and compact, and the materials had obtained better thermal and mechanical properties. This research provided a novel and environment-friendly method for the modification technology of keratin, and it was beneficial to expand the application fields of biomass materials.
... For the study of intramolecular disulfide bonds, spectroscopy techniques such as Raman or IR have been applied. 40,41 Also as we reported previously, we conducted Raman tests on GERTs and observed that for GERTs with multilayered BDT, two new Raman bands appeared at around 380 and 520 cm −1 , which were attributed to the disulfide bond. 20 This further confirms the interlayer bonding of embedded BDT. ...
The plasmonic molecular junctions are of wide interest for the studies of physics, chemistry and materials science. For the three-dimensional (3D) molecular junctions inside plasmonic core-shell nanoparticles, the junction morphology plays a vital role in optical properties and related applications. However, the atomistic insights into the structural evolutions of interior 3D molecular junctions are still lacking. This work investigated the formation of 3D molecular junctions in gap-enhanced Raman tags (GERTs) experimentally and theoretically. With monolayered or double-layered molecules embedded, either symmetric or asymmetric 3D junctions can be tailored in experiments. By studying the interactions between Au atoms and thiolated molecules using molecular dynamics simulation, we revealed that the junction morphology was determined by the embedded molecular layers. Asymmetric nanogaps were formed due to different molecular orientations, packing and ring stacking structures. We further designed a multi-nucleation strategy to obtain symmetric and uniform junctions with multi-layered molecules. This work offers a general framework for the simulations of complex junction systems, brings new insights into the morphology evolution of 3D plasmonic junctions, and facilitates the developments of molecular-based plasmonic nanodevices.
... www.nature.com/scientificreports/ absorbance could not be collected at wavenumbers below ~700 cm −1 with the ATR set-up used in this study 71,74 . The FTIR data show increased absorbances for both C-O bonding and glycosylation, providing evidence for this increased intramolecular bonding as a result of Ca 2+ interactions with PGM. ...
Mucus is responsible for controlling transport and barrier function in biological systems, and its properties can be significantly affected by compositional and environmental changes. In this study, the impacts of pH and CaCl2 were examined on the solution-to-gel transition of mucin, the primary structural component of mucus. Microscale structural changes were correlated with macroscale viscoelastic behavior as a function of pH and calcium addition using rheology, dynamic light scattering, zeta potential, surface tension, and FTIR spectroscopic characterization. Mucin solutions transitioned from solution to gel behavior between pH 4–5 and correspondingly displayed a more than ten-fold increase in viscoelastic moduli. Addition of CaCl2 increased the sol-gel transition pH value to ca. 6, with a twofold increase in loss moduli at low frequencies and ten-fold increase in storage modulus. Changing the ionic conditions—specifically [H⁺] and [Ca²⁺] —modulated the sol-gel transition pH, isoelectric point, and viscoelastic properties due to reversible conformational changes with mucin forming a network structure via non-covalent cross-links between mucin chains.
... The unfolding of zein in aqueous HAuCl 4 solution in the presence of surfactant leads to an exposure of cysteine which initiate the reduction of Au 3+ to Au 0 . [31][32][33] This result can be correlated to the change in the secondary structure of zein protein when functionalized on AuNPs. 34 To elaborate further, the CD measurement of zein by varying the temperature from 20 C to 90 C suggested that zein denatured in the range of 65 C to 80 C (Fig. S3a †). ...
In this article, we report a simple method to synthesize biodegradable zein films functionalized with gold nanoparticles (AuNPs) with significantly improved mechanical properties, as an environmentally benign substitute to biologically hazardous polymers. Zein-coated AuNPs were synthesized using the zein protein as a reducing agent and characterized with IR, UV, CD, ζ-potential, and TEM measurements. The zein protein interaction with the negatively charged surface of AuNPs provides excellent strength to the zein thin film. For the first time, FT-IR spectral studies suggested the strong interaction between AuNPs and zein protein, which was further supported by the higher binding constant (K b) value. The films were characterized for mechanical properties with spectroscopic and physical experimental investigations. The surface morphology of AuNP-doped zein film was explored by AFM and SEM, which suggested that the AuNPs prevent the buckling of zein film and increase the strength as well as flexibility of the film.
... This is another evidence that the formation of the HN þ group in the thiol derivative was performed by the transfer of SH proton to the azomethine nitrogen. On the other hand, the spectrum of 2 displayed a band due to C]N frequency at almost the same position as the thiol monomer 1, Fig. 1 the SeS bond stretching frequency usually occurred in the range 500e540 cm À1 as a weak band [18,19]. The present compound exhibited a weak band in its IR spectrum at 550 cm À1 , which could be assigned to the SeS stretching frequency, Fig. 1. ...
Facile conversion of a thiol-Schiff base (obtained from the condensation of 2-hydroxynaphthaldehyde and 2-aminothiophenol) to the corresponding homodisulfide derivative is described. The molecular structure and stoichiometry of the thiol (1) and disulfide (2) compounds were characterized by analytical and spectroscopic techniques. The structure of the disulfide derivative was also confirmed by single crystal X-ray analysis. Theoretical calculations based on density functional theory (DFT) were established to verify the structures of the thiol in both solid and solution forms as well the structure of the disulfide. The global chemical reactivity descriptors were estimated from the energy of the HOMO and LUMO orbitals.
... Most notably, the bond around 509 cm −1 changed after treatment. This signal corresponds to the disulfide bonds of RNaseA and while the bond only lost a slight amount of total intensity, the changes of the three underlying signals indicated a shift in disulfide bond conformation as described in ref. [50] Furthermore, changes at the C─S bond around 645 cm −1 and the increase of the SO signal at 1047 cm −1 indicated the oxidation of the sulfur-containing amino acids methionine and cysteine. Modifications of sulfur-carrying amino acids by both lowpressure and atmospheric pressure plasmas have already FIGURE 4 Relative RNaseA activity after VHF-CCP treatment: Enzyme activity was measured after either treatment with vaporized water or hydrogen peroxide (panel A) or after treatment with hydrogen plasma alone or in combination with vaporized water or hydrogen peroxide (panel B). ...
Low-pressure plasmas are a promising alternative to modern sterilization processes. As plasma is a surface process, multilayered stacks of spores are a crucial challenge to overcome. Here, a combined process of condensed hydrogen peroxide and hydrogen plasma is analyzed for its efficacy against various spore concentrations showing a clear increase in efficacy using a combined process compared to the two steps used separately. Besides spores, protein contaminations are a major issue in clinics and the combined process is investigated for protein removal efficiency using the well-established BSA model. Furthermore, RNase A serves as a difficult-to-inactivate protein model to investigate protein inactivation efficiency. Finally, inactivation mechanisms of RNase A with a special focus on sulfur-based modifications are investigated using Raman spectroscopy.
... Because prior results with SP-B peptides [17,18,58,63,79,80] suggested that the N-terminal sequence (i.e., residues 1-7) plays key roles in the surfactant properties of full-length SP-B, we next engineered the Super Mini-B (S-MB), a 41-residue peptide mimic in which residues 1-7 was covalently attached to the N-terminus of MB [S-MB (residues 1-41): FPIPLPYCWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRC]. S-MB might be expected to show higher surfactant activity than MB, because the former peptide may more accurately mimic the leaflet structure containing the N-and C-terminal domains of SP-B (Figure 1). ...
Surfactant protein (SP)-B is a 79-residue polypeptide crucial for the biophysical and physiological function of endogenous lung surfactant. SP-B is a member of the saposin or saposin-like proteins (SAPLIP) family of proteins that share an overall three-dimensional folding pattern based on secondary structures and disulfide connectivity and exhibit a wide diversity of biological functions. Here, we review the synthesis, molecular biophysics and activity of synthetic analogs of saposin proteins designed to mimic those interactions of the parent proteins with lipids that enhance interfacial activity. Saposin proteins generally interact with target lipids as either monomers or multimers via well-defined amphipathic helices, flexible hinge domains, and insertion sequences. Based on the known 3D-structural motif for the saposin family, we show how bioengineering techniques may be used to develop minimal peptide constructs that maintain desirable structural properties and activities in biomedical applications. One important application is the molecular design, synthesis and activity of Saposin mimics based on the SP-B structure. Synthetic lung surfactants containing active SP-B analogs may be potentially useful in treating diseases of surfactant deficiency or dysfunction including the neonatal respiratory distress syndrome and acute lung injury/acute respiratory distress syndrome.
... Analysis of all the Raman disulfide peaks revealed a strong dependence of S-S wavenumber position on the torsional angle w(C-S-S-C) which provides information about the internal rotation about C-S and C-C bonds in C a -C b -S-S-C b 0 -C a 0 conformations. 86,87 The peak at 510 AE 5 cm À1 (B507 cm À1 , in our case) is suggestive of the gauche-gauche-gauche conformation (w: AE601) that was confirmed by careful analysis of the disulfides in the crystal structure using PyMol (Fig. 1A). The additional peaks observed at 525 AE 5 cm À1 and at 540 AE 5 cm À1 are attributed to gauche-gauche-trans (w: AE80-901) and trans-gauche-trans conformations, respectively. ...
The self-assembly of proteins triggered by a conformational switch into highly ordered β-sheet rich amyloid fibrils has captivated a burgeoning interest in recent years due to the involvement of amyloids in a variety of human diseases and a diverse range of biological functions. Here, we have investigated the mechanism of fibrillogenesis of human serum albumin (HSA), an all-α-helical protein, using an array of biophysical tools that include steady-state as well as time-resolved fluorescence, circular dichroism and Raman spectroscopy in conjunction with atomic force microscopy (AFM). Investigations into the temporal evolution of nanoscale morphology using AFM revealed the presence of ring-like intermediates that subsequently transformed into worm-like fibrils presumably by a ring-opening mechanism. Additionally, a multitude of morphologically-diverse oligomers were observed on the pathway to amyloid formation. Kinetic analysis using multiple structural probes in-tandem indicated that HSA amyloid assembly is a concerted process encompassing a major structural change that is primarily mediated by hydrophobic interactions between thermally-induced disordered segments originating in various domains. A slower growth kinetics of aggregates suggested that the protein structural reorganization is a prerequisite for the fibril formation. Moreover, time-dependent Raman spectroscopic studies of HSA aggregation provided key molecular insights into the conformational transitions occurring within the protein amide backbone and at the residue-specific level. Our data revealed the emergence of conformationally-diverse disulfides as a consequence of structural reorganization and sequestration of tyrosines into the hydrophobic amyloid core comprising antiparallel cross β-sheets.
... We investigated the specific interaction of the protein with the AuNP and AgNP using Raman spectroscopy which states that the disulfide groups present on the protein have an important role in the conjugation with laser-generated Au and Ag nanoparticle. The peak centered at 513 cm -1 corresponds to the disulfide stretching mode of protein (Chen and Lord 1976;Nakamura et al. 1997;Biswas et al. 2007). Raman analysis reveals that the intensity of the disulfide stretch at 513 cm -1 is greatly reduced in the conjugated S-ovalbumin. ...
Pure gold and silver nanoparticle (NP)
generation and their conjugation with protein S-ovalbumin
using in situ conjugation process have been
reported. The in situ conjugation involves nanosecond
pulse laser ablation of pure metal target in the protein
S-ovalbumin solution. Transmission electron microscopy
(TEM) and UV–Visible absorption results
show decrease in mean NP size along with narrow
particle size distribution on ablation in S-ovalbumin
solution as compared to ablation in water for both Au
and Ag NPs. Also, the NP size reduction was found to
be dependent on the concentration of S-ovalbumin.
For AuNPs, spherical NPs of mean size 4 nm with
particle size distribution 2–6 nm were obtained at
300 nM S-ovalbumin concentration. Further, it has
been observed that the resultant in situ-conjugated
colloid gold and silver NP solutions were quite stable
even in the presence of NaCl at physiological salt
concentration (0.15 M). On post-laser irradiation
(532 nm, 15 mJ) for 20 min, 9 nm red shift in surface
plasmon resonance peak (SPR), along with increased
broadening towards longer wavelength, was observed
in the AuNPs–S-ovalbumin sample. Further increase
in the time of irradiation showed shift in AuNPs–Sovalbumin
SPR towards lower wavelength. On laser
irradiation (532 nm, 15 mJ) for 20 min, no significant
change was observed in the line shape of the plasmon
absorption band of the AgNPs–S-ovalbumin conjugate.
FTIR spectra revealed that S-ovalbumin peptide
backbone and secondary structure remain unchanged
on laser irradiation during in situ conjugation process.
Thus, integrity of S-ovalbumin does not get affected,
and no degradation of S-ovalbumin takes place on
laser-induced in situ conjugation. Raman results
confirm that both Au and Ag NPs interact with
S-ovalbumin via thiol-bearing cysteine residues of the
disulfide bond.
... Peak assignments for the Raman spectra shown in Fig. 6 are summarized in the Table 1 [20,26,27]. ...
Partially oxidized spherical silver nanoparticles (AgNPs) of different size are prepared by pulsed laser ablation in water and directly conjugated to protein S-ovalbumin for the first time and characterized by various optical techniques. UV-Visible spectrum of AgNPs showed localized surface plasmon resonance (LSPR) peak at 396 nm which red shift after protein addition. Further the increased concentration of AgNPs resulted a decrease in intensity and broadening of S-ovalbumin peak (278 nm), which can be related to the formation of protein NPs complex caused by the partial adsorption of S-ovalbumin on the surface of AgNPs. The red shift in LSPR peak of AgNPs after mixing with S-ovalbumin and decrease in protein-characteristic peak with increased silver loading confirmed the formation of protein-AgNPs bioconjugates. The effect of laser fluence on the size of AgNPs and nanoparticle-protein conjugation in the size range 5-38 nm is systematically studied. Raman spectra reveal broken disulphide bonds in the conjugated protein and formation of Ag-S bonds on the nanoparticle surface. Fluorescence spectroscopy showed quenching in fluorescence emission intensity of tryptophan residue of S-ovalbumin due to energy transfer from tryptophan moieties of albumin to AgNPs. Besides this, small blue shift in emission peak is also noticed in presence of AgNPs, which might be due to complex formation between protein and nanoparticles. The binding constant (K) and the number of binding sites (n) between AgNPs and S-ovalbumin have been found to be 0.006 M-1 and 7.11, respectively.
... The conformation of C-S-S-C group in a protein is in equilibrium of gauche and trans for each bond. [59,60] The gauchegauche-gauche conformation is the most stable with minimum energy. When one of the gauche becomes trans, the stability of the structure decreases. ...
The mechanical properties of the Stratum Corneum (SC) have been studied by different authors at the macroscopic level, but the modification of its ultra structure during mechanical extension remains unknown. Moreover, little is described about the effect of the mechanical stress on SC barrier function. In this study, we have examined the SC structure changes, at the molecular level, during uniaxial tensile experiments. This was performed on isolated SC samples using Raman spectroscopy. We could identify the strain status of the analyzed samples by using combination of Raman spectra and Partial Least Squares processing. In addition, this approach provided information about lipids and proteins behavior during the sample extension. The structure of the intercellular lipids bilayer became less organized up to ~9% deformation. For higher strains, a plateau corresponding to the minimum organization is observed till the complete failure of the sample. In the same time, protein structures including desmosomes, were characterized by monotonic secondary structure modifications for deformations up to ~9% followed by a plateau. These observations are relevantly demonstrating the effect of extension on the skin barrier state. Such an approach could be objectively used for clinical applications to evaluate skin discomfort degree and skin elastic behavior. This could therefore help with proof of efficacy for cosmetic and dermatologic products. Copyright © 2013 John Wiley & Sons, Ltd.
... Raman spectroscopy is being currently used also to characterize lipids [40] and phospholipids [41,42] at the air-water interface. This method has been useful to get information about order and tilt angle of hydrophobic chains at the air-water interface and to investigate the interaction of AMP with phospholipid mixtures [43] and, also recently, to investigate aminoacids [44], the disulfide bond in peptides [45] and phospholipid bilayers on solid substrates [46]. An excellent description of this technique used for the study of nucleic acids, viruses, and proteins has been reported by Blanch et. ...
... The conformer of S-MB at 49.8 nsec was selected to model the homodimer because its conformation was closest to the b-sheet structure for residues Tyr- 7 to Arg-12 as predicted from the PASTA and AGGRESCAN programs (see Results). Two S-MB monomers were initially docked to form a homodimer using ZDOCK [73], similar to that previously described for the docking of the N-terminal domain of SP-B [74]. The lowest energy conformer of this initial SMB homodimeric structure was then further refined by using RosettaDock (www.rosettacommons.org), ...
Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.
FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.
Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.
Vibrational spectroscopy methods display great potential in the study of biomolecules. The complexity of these molecular systems, though, is often a hindrance for the interpretation of the experimental spectra. Selective techniques should therefore be improved and applied extensively. This article presents surface enhanced Raman spectra of an important model protein, namely, wheat germ agglutinin. To obtain these spectra, a novel substrate for the protein has been developed. The substrate is based on gold nanospheroids, produced by wet synthesis. The nanoparticles are then functionalized by reaction with a solution of β-D-thioglucose to increase the affinity of the agglutinin for the surface. When nanoparticle films are placed in contact with diluted agglutinin solutions, vibrational bands of the protein appear and can be easily discriminated from those of the substrate. The Raman bands of the substrate and of the protein have then been assigned correlatively. On this basis, we conclude that the interaction between the agglutinin and the nanoparticles is selectively detected only when the metal surface is suitably functionalized.
The methyl-nitro-pyridine-disulfide derivative [2,2′-disulfanodiylbis(6-metyl-3-nitropyridine)] was synthesized and characterized by means of structural and spectroscopic measurements. On the basis of X-ray diffraction studies, it was found that the studied compound crystallizes in the centrosymmetric monoclinic space group P21/n (Z=2). The disulfide C-S-S-C bridge links two identical fragments formed by pyridine rings substituted with methyl and nitro groups. Such a structure was confirmed by ¹H and ¹³C NMR studies as well as IR, Raman, UV-VIS and emission spectra. Quantum chemical DFT calculations were applied in the analysis of the obtained results. The vibrational characteristics were reported and dynamical properties of this moiety were discussed. A full set of the normal modes characteristic for the disulfide bridge was identified and assigned to the respective IR and Raman bands. The results of structural and spectroscopic studies were used to find the dependence between the conformation of the ϕ-S-S-ϕ system and its optic properties. The experimental electron and emission spectra were analyzed in terms of the calculated singlet and triplet states that allowed assigning the unique spectral pattern originating from the electrons of the C-S-S-C bridge system.
Background: Glyco disulfide gold nanoparticles (GDAuNPs) were prepared by three methods: direct, photochemical irradiation and ligand substitution. Glyco disulfide acted as reducing and capping agents of gold ions, to produce AuNPs GD1–GD16. Results: Shorter chains of glyco disulfides (n = 1 and 2) offered monodispersed and stable GDAuNPs in physiological pH, while longer chains (n = 3) furnished unstable nanoparticles. ζ-potential study of direct method GDAuNPs revealed surface charge dependency on the alkyl unit length. Transmission electron microscope imaging indicated that sizes/shapes of the ligand exchange AuNPs remained post-exchange step. The mechanism of GDAuNP formation was forecast as the Ostwald ripening effect at low pH of ligand (5.1-8.9) and reinforcement of static stabilization at high pH (12.4–13.0). Conclusion: GDAuNPs recorded moderately anticancer activity against the A549 cancer cell line, with IC 50 between 14.95 and 64.95 μg/ml.
The shape of wool yarns was changed by laccase-assisted grafting of tyrosine. Prior to tyrosine grafting a cysteine pre-treatment was optimized aiming to increase the amount of thiol reaction groups available. The best operational conditions for laccase-assisted tyrosine grafting were: i) pre-treatment with cysteine (2.2 mM) in a solution of 20% ethanol, 15% propylene glycol and 0.5% benzyl alcohol, pH = 10, 40 °C; ii) tyrosine grafting with 3.0 mM tyrosine, 18 U/mL laccase, pH = 5, 40 °C. The shape modification was evaluated by number of curly twists determination on the grafted yarn samples. The thermal and mechanical properties of the grafted wool yarns was evaluated by TGA, DSC and breaking strength determination. The amount of free thiols and weight gain were assessed aiming to infer the role of the cysteine pre-treatment on the final tyrosine grafting and shape modification. The laccase-assisted grafting of tyrosine onto wool yarns have influenced the thermal and mechanical properties of the yarns however without compromising their structural integrity for the final application purposes. The developed methodology to impart new shape to wool yarns is presented herein as an environmentally friendly alternative to chemical methods. The new findings revealed great potentialities for application in similar fibers like hair.
Chitosan exhibits great versatility in various biomedical fields and mesoporous silica nanoparticles have emerged as an interesting material in biomedical areas owing to their outstanding physio-chemical properties. The combination of inorganic silica and organic polymer such as chitosan, make them suitable for a wide range of biomedical applications. Here, we have explored the benefits of chitosan and silica by synthesizing chitosan-silica nanohybrid. In the synthesis of chitosan-silica (CS–Si) nanohybrid, chitosan is modified by thioglycolic acid and mesoporous silica MCM-41(Mobil Composition of Matter number 41) is functionalized by 3-(trimethoxysilyl)-1-propane thiol (TMSP). The modified chitosan and thiol functionalized MCM-41(inorganic network) is then linked through disulfide bond by oxidation process or oxidative coupling, resulting in the formation of inorganic-organic hybrid material. The hybrid material was characterized by FTIR, Raman, XRD, TGA, Zeta potential, EDX, Proton NMR and SEM techniques. The antibacterial results indicated that gram-negative (E. coli) bacteria exhibit better inhibition zone than gram-positive (B. subtilis) bacteria. The DPPH scavenging capability of synthesized hybrid was found to be 68%. The drug (quercetin) encapsulation efficiency of hybrid material was calculated to be 92.38% and more drug releases in acidic medium (pH 5.0) than at pH 7.4, so we can conclude that hybrid material shows pH-dependent drug releasing behavior. The results show that synthesized nano-hybrid material possess good antibacterial and antioxidant activities and is also a good nanocarrier for drug delivery application.
The understanding of progressive advances in the development of intelligent systems based on biocompatible, biodegradable, and renewable resources and their applications in important fields, such as medical, pharmaceutical, agricultural, and industrial, have increased attention in the use of cellulose for obtaining stimuli-responsive materials. Within the smart polymer systems, one of the most relevant is hydrogels, which are materials with important characteristics of water absorption, elasticity, and multifunctionality. These materials have been developed from several renewable sources such as chitin, starch, and cellulose; in particular, cellulose and cellulose by-products have allowed the designing of hydrogels with a wide range of functionalities and applications. Of interest for us, hydrogels are capable of experiencing a response as a result of changes in their environment or external stimuli, which can be chemical or/and physical, for example, pH, temperature, ionic strength, and electromagnetic fields, among others. Initially, in this chapter, physical-chemical properties and the importance of cellulose as a polymeric material will be described, emphasizing its use for obtaining stimuli-responsive hydrogels and their applications, in particular, in the biomedical field. In this way, their properties, the obtaining routes, and characterization methods of stimuli-responsive cellulose-based hydrogels (CBHG), from physical to chemical, are analyzed and described. Finally, significant applications of stimuli-responsive, CBHGs in drug-controlled release and tissue engineering are shown and discussed.
Cerastoderma glaucum, a marine bivalve inhabiting Lake Timsah, is surrounded by different pathogenic organisms. The present study evaluates the antimicrobial activities, cytotoxicity and characterization of C. glaucum extracts. Chloroform, methanol and acidic tissue extracts were prepared from C. glaucum collected during winter and summer seasons. Winter acidic extract exhibits potent antimicrobial activities against 21 bacterial, 2 yeast and 2 viral strains. The inhibition zone of this extract ranges from 10 mm against Klebsiella oxytoca, Pseudomonas stutzeri, Globicatella sulfidifaciens and Bacillus (B. badius, B. amyloliquefaciens and B. pumilus) to 24 mm against Shigella flexneri. Also, the inhibition viral activities of this extract at a concentration of 62.5 µg ml−1 against Hepatitis A virus and Herpes simplex virus type 1 (HSV-1) are 62.383% and 57.035%, respectively, with low cytotoxicity of 24.030%. Furthermore, winter acidic extract of C. glaucum has the highest total protein contents (9.8 mg ml−1) compared with the other extracts. Moreover, the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) indicates the presence of four clear low molecular weight peptides bands; 8.588, 7.237, 4.423 and 2.692 kDa. Fourier transform-infrared (FT-IR) analysis indicates the presence of 12 functional groups of proteins in winter acidic extract of C. glaucum at appropriate wavelengths.
Composites of poly (vinylidene fluoride – co – hexafluoro propylene) (PVdF – HFP) incorporating 10 wt. % Bis(trifluoromethane)sulfonimide lithium salt (LiTFSI) and 10 wt. % particles of nanoparticulate silica (nm-SiO2), nanoparticulate titania (nm-TiO2) and fumed silica (f-SiO2) were prepared by electrospinning. These membranes served as host matrix for the preparation of composite polymer electrolytes (CPEs) following activation with lithium sulfur battery electrolyte comprising 50/50 vol% Dioxolane/Dimethoxyethane with 1 M LiTFSI and 0.1 M LiNO3. The membranes consist of layers of fibers with average fiber diameter of 1–2 μm. CPEs with f-SiO2 exhibited higher ionic conductivity with a maximum of 1.3 × 10−3 S cm−1 at 25 °C obtained with 10 wt. % filler compositions. The optimum CPE based on PVdF – HFP with 10 wt% f-SiO2 exhibited enhanced charge–discharge performance in Li – S cells at room temperature eliminating polysulfide migration, delivering initial specific capacity of 895 mAh g−1 at 0.1 C-rate and a very low electrolyte/sulfur (E/S) ratios between 3:1 to 4:1 ml.g-1. The CPEs also exhibited very stable cycling behavior well over 100 cycles (fade rate ~0.056%/cycle), demonstrating their suitability for Li – S battery applications. In addition, the interconnected morphological features of PVdF – HFP result in superior mechanical properties (200-350% higher tensile strength). Higher Li-ion conductivity, higher liquid electrolyte uptake (>250%) with dimensional stability, lower interfacial resistance and higher electrochemical stability are some of the attractive attributes witnessed with these CPEs. With these improved performance characteristics, the PVdF – HFP system is projected herein as suitable polymer electrolytes for high-performance Li – S rechargeable batteries.
The disproportionation of polysulfide (PS) is a long-neglected and vital issue that causes the fast capacity fading of Li–S batteries. Based on the hard and soft acids and bases (HSAB) theory, a large size N-methyl-N-ethyl pyrrolidinium (MEP⁺) cation is proposed to complex and stabilize the PS in electrolyte. The disproportionation of PS is successfully suppressed by this simple method, thereby avoiding the precipitation of sulfur in the electrolyte and reducing the loss of the active materials. The mutual interaction mechanism between MEP⁺ and Sn²⁻ in electrolyte is comprehensively investigated and verified for the first time, via both density functional theory (DFT) calculation and experimental characterization. It enables the 5000 mA h Li–S batteries (soft package type) to achieve initial specific energy over 300 Wh kg⁻¹ and maintain over 65% after 100 charge/discharge cycles at 1/20 C, while merely 24% is remained at 59 cycles without MEP⁺. This interesting finding is believed to shed light on the further development of Li–S batteries.
Extracted from the chloroplasts of green plants, Photosystem I (PSI) is a multi-subunit protein complex that can be deposited as a thin film onto electrodes and incorporated into biohybrid photovoltaic devices to convert sunlight into usable energy. Previously, our group characterized persistent photocurrents over nine months using PSI multilayer films in an aqueous solution of redox mediators. However, the loss of periphery PSI proteins upon exposure to mediator solutions is known to affect the photocatalytic performance of the overall film. The cross-linker 2-iminothiolane (2IT) is capable of connecting protein molecules with covalent bonds, and is herein used to stabilize PSI multilayer films on gold substrates against significant desorption/degradation. Results showed that the thickness retainment of the cross-linked PSI films was superior to that of non-cross-linked films. PSI films modified by 2IT maintained production of photocurrent with long-term exposure to aqueous solutions in contrast to non-cross-linked control films.
Reduction-responsive liposome was prepared by incorporating benzyl disulfide in egg phosphatidylcholine (egg PC) liposomal bilayer. The fluorescence quenching degree of calcein enveloped in egg PC liposome bearing benzyl disulfide decreased from 62.7% to 49.2% and the mean hydrodynamic diameter of the liposome decreased from 276 nm to 206 nm when the egg PC to benzyl disulfide weight ratio increased from 20:0 to 20:10. The liposomes bearing benzyl disulfide were multi-lamellar vesicles on TEM photos. It was confirmed by Raman spectroscopy that benzyl disulfide was loaded in the liposomal bilayer. The release degree of calcein enveloped in liposome incorporating benzyl disulfide was investigated for 24 h at 25 °C, 37 °C, and 45 °C, when the concentration of dl-dithiothreitol (DTT) was 0 mM, 5 mM, 10 mM and 30 mM. At all the DTT concentrations tested, the release degree was more extensive as the temperature was higher. At all the temperatures tested, the release degree became more extensive as the DTT concentration increased. For example, the maximum release degree at 37 °C of calcein loaded in liposome of which egg PC to benzyl disulfide weight ratio was 20:5 increased from 2.9% to 14.8% when DTT concentration increased from 0 mM to 10 mM. DTT could reduce benzyl disulfide to benzyl thiols and the fragments of benzyl disulfide would re-orient due to its polarity change in the liposomal membrane, resulting in the membrane fluctuation and the promoted release. At all the temperatures and all the DTT concentrations tested, the release was more sensitive to the DTT concentration as the benzyl disulfide content in liposome was higher, indicating that the reduction of the disulfide compound was responsible for the promoted release.
Background:
In vivo Raman spectroscopy is a powerful tool for real-time analysis and in situ evaluation of tissues such as the skin. The efficiency of this technique has been widely demonstrated as a label-free method for in vivo evaluation of the skin. The aim of this study is to gather information about inter- and intra-individual variations in the spectral descriptors of water content and structure, organization of the lipid barrier and structure of proteins in the stratum corneum (SC).
Methods:
In vivo SC measurements were performed on 17 female volunteers aged 20-30 years (phototypes I and II). For intra-individual variability, spectral collection was performed on 5 successive days per volunteer. Shapiro-Wilk and Cochran tests were applied to check the normality and the homoscedasticity of variances. ANOVA was then applied to evaluate intra- and intergroup variability.
Results:
ANOVA was performed on the spectral descriptors of water content and structure, organization of the lipid barrier and secondary structure of proteins in the SC. No significant intra- and interday variability was observed for all volunteers. Despite the low value of the total relative standard deviation, a highly significant variation was observed between volunteers.
Conclusion:
Interindividual variability for Raman measurements is significant for a set of volunteers with normal nondiseased SC and close phototypes. This variability should be taken into consideration as a threshold for significant variance when working in vivo.
The skin is the largest organ of the human body, accounting for ~10% of the body weight. The quality of its state and functionality is essential for the human health. Dry skin is a common phenomenon in various physiopathological dysfunctions. The uppermost layer of the skin, the stratum corneum assumes the first barrier between organism and environment, it is thus considered as the main element controlling skin hydration. This work, combining technical and methodological developments, led to highlight the molecular mechanisms involved in skin dryness phenomena by confocal Raman microspectroscopy. The molecular link between hydration and mechanical stress of SC ex vivo is described in detail involving lipids and proteins. This work has also focused on the characterization of supramolecular changes related to the deformations of the SC under mechanical stress. In parallel, this work illustrates the effectiveness of vibrational spectroscopy for evaluation of moisturizers mechanisms of action. The non-invasive nature of Raman spectroscopy allowed exploiting the high potential of this technique by transposing spectral descriptors obtained ex vivo to in vivo. Thus, we developed an in vivo approach coupling Raman spectroscopy and partial least squares method (PLS) for indirect quantification of different physico-chemical and functional parameters including the SC lipids and water leading to an overall characterization of the SC physiopathological status.
A family of peptoid dimers developed to mimic SP‐B is presented, where two amphipathic, cationic helices are linked by an achiral octameric chain. SP‐B is a vital therapeutic protein in lung surfactant replacement therapy, but its large‐scale isolation or chemical synthesis is impractical. Enhanced biomimicry of SP‐B's disulfide‐bonded structure has been previously attempted via disulfide‐mediated dimerization of SP‐B 1‐25 and other peptide mimics, which improved surface activity relative to the monomers. Herein, the effects of disulfide‐ or “click”‐mediated (1,3‐dipolar cycloaddition) dimerization, as well as linker chemistry, on the lipid‐associated surfactant activity of a peptoid monomer are described. Results revealed that the ‘clicked’ peptoid dimer enhanced in vitro surface activity in a DPPC:POPG:PA lipid film relative to its disulfide‐bonded and monomeric counterparts in both surface balance and pulsating bubble surfactometry studies. On the pulsating bubble surfactometer, the film containing the “clicked” peptoid dimer outperformed all presented peptoid monomers and dimers, and two SP‐B derived peptides, attaining an adsorbed surface tension of 22 mN m ⁻¹ , and maximum and minimum cycling values of 42 mN m ⁻¹ and near‐zero, respectively. © 2009 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 538–553, 2009.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
The Arg14 to Cys (R14C) mutation in the human gammaD-crystallin (HGD) gene has been associated with a juvenile-onset hereditary cataract. We showed previously [Pande, A., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1993-1998] that rapid oxidation of Cys14 in the mutant leads to the formation of intermolecular, disulfide-cross-linked aggregates at physiological pH. Here we present a Raman spectroscopic analysis of R14C and HGD and show that R14C forms such aggregates even at pH 4.5. The lower pH enabled us to monitor the evolution of a variety of disulfide cross-links with distinct conformations around the CC-SS-CC dihedral angles. At least three cysteine residues are involved, forming protein-protein cross-links through disulfide-exchange reactions. From the pattern of the S-S and Trp Raman bands, we infer that Cys32 is likely to be involved in the cross-linking. The data suggest that protein precipitation in the mutant may not be the direct result of disulfide cross-linking, although such cross-linking is the initiating event. Thus, our Raman data not only enhance the understanding of the reactivity of Cys14 in the R14C mutant and the mechanism of opacity, but also shed light on the mechanism of oxidative degradation during long-term storage of thiol-containing pharmaceuticals.
Protein-nanoparticle interactions are of central importance in the biomedical applications of nanoparticles, as well as in the growing biosafety concerns of nanomaterials. We observe that gold nanoparticles initiate protein aggregation at physiological pH, resulting in the formation of extended, amorphous protein-nanoparticle assemblies, accompanied by large protein aggregates without embedded nanoparticles. Proteins at the Au nanoparticle surface are observed to be partially unfolded; these nanoparticle-induced misfolded proteins likely catalyze the observed aggregate formation and growth.
Pulmonary surfactant is a lipid-protein complex that coats the interior of the alveoli and enables the lungs to function properly. Upon its synthesis, lung surfactant adsorbs at the interface between the air and the hypophase, a capillary aqueous layer covering the alveoli. By lowering and modulating surface tension during breathing, lung surfactant reduces respiratory work of expansion, and stabilises alveoli against collapse during expiration. Pulmonary surfactant deficiency, or dysfunction, contributes to several respiratory pathologies, such as infant respiratory distress syndrome (IRDS) in premature neonates, and acute respiratory distress syndrome (ARDS) in children and adults. The main clinical exogenous surfactants currently in use to treat some of these pathologies are essentially organic extracts obtained from animal lungs. Although very efficient, natural surfactants bear serious defects: i) they could vary in composition from batch to batch; ii) their production involves relatively high costs, and sources are limited; and iii) they carry a potential risk of transmission of animal infectious agents and the possibility of immunological reaction. All these caveats justify the necessity for a highly controlled synthetic material. In the present review the efforts aimed at new surfactant development, including the modification of existing exogenous surfactants by adding molecules that can enhance their activity, and the progress achieved in the production of completely new preparations, are discussed.
Sea anemone contain a number of closely related peptide heart stimulants. In the present investigation, the conformation of anthropleurin A from Anthopleura xanthogrammica was investigated by laser Raman, circular dichroism, and fluorescence spectral methods and by the Chou-Fasman method using sequence data. The recent 13C NMR data of the peptide (Norton, R.S., and Norton, T.R. (1979) J. Biol. Chem., in press) provided useful information for the interpretation of the above-mentioned spectral data. The results from these spectral methods suggested that anthropleurin A and the related sea anemone peptides are roughly spherical in shape due to the presence of some beta-bends, possibly due to a beta-pleated sheet region and due to the 3 cystine residues in the peptide which exist in the gauche-gauche-gauche configuration. The sole tyrosine residue is exposed to the solvent, a finding which has now been confirmed by 13C NMR. The laser Raman and fluorescence spectral procedures showed that one or more of the tryptophan residues are buried. Interestingly, the reduction of the native protein with dithioerythritol did not change the spherical shape even in the presence of 5 M guanidine HCl and the carboxymethylcysteine derivative of the peptide was folded even in the presence of the denaturing agent, guanidine HCl.
Laser Raman spectroscopy is used to probe the heterogeneous substructure of the large contractile protein myosin. Some peaks are assigned to specific chemical groups of the molecule; others, notably the conformationally sensitive amide III vibrations, provide information on the structurally distinct regions of the molecule. Deuteration of the NH groups is instrumental in the assignment of these vibrational modes. The relative intensities of bands typical of alpha-helical conformations (near 1265 and 1304 cm-1) and bands associated with nonhelical structure (near 1244 cm-1) are sensitive indicators of myosin substructure and represent potentially useful probes of conformational changes.
Pulmonary surfactant contains two hydrophobic polypeptides, SP-B and SP-C, with known amino acid sequences and with truncated subforms lacking the N-terminal residues. Treatment of SP-C with KOH releases fatty acids (palmitic acid to more than 85%) in molar ratios of 1.8-2.0 relative to the polypeptide. Furthermore, plasma-desorption mass spectrometry shows native SP-C of both the intact and truncated types to be monomers with masses about 500 units higher than those expected for the polypeptide chains. After treatment with KOH, trimethylamine, or dithioerythritol, the polypeptide masses are obtained. These results prove that native SP-C is a lipopeptide with two palmitoyl groups covalently linked to the polypeptide chain. The deacylation conditions, the presence of two cysteine residues in the polypeptide, and the absence of other possible attachment sites establish that the palmitoyl groups are thioester-linked to the two adjacent cysteine residues. In contrast, the major form of porcine SP-B is a dimer without fatty acid components. That SP-C is a true lipopeptide with covalently bound palmitoyl groups suggests possibilities for functional interactions. It gives a direct physical link between SP-C and surfactant phospholipid components. Long-chain acylation may constitute a means for association of proteins with membranes and could conceivably modulate the stability and biological activity of surfactant films.
Hydrophobic surfactant-associated protein of Mr 6000-14,000 was isolated from ether/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of Mr 6000-14,000 in immunoblot analysis and was used to screen a lambda gt11 expression library constructed from adult human lung poly(A)+ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused beta-galactosidase-SPL(Phe) gene in Escherichia coli yielded an immunoreactive Mr 34,000 fusion peptide. Hybrid-arrested translation with this cDNA and immunoprecipitation of [35S]methionine-labeled in vitro translation products of human poly(A)+ RNA with a surfactant polyclonal antibody resulted in identification of a Mr 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. The larger RNA and translation product indicates that SPL(Phe) is derived by proteolysis of a large polypeptide precursor. The amino acid sequence of the predicted protein, beginning Phe-Pro-Ile-Pro-Leu-Pro-Try-, comprises a hydrophobic peptide that is a major protein component of surfactant lipid extracts used successfully to treat hyaline membrane disease in newborn infants. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.
Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.
This article starts with a brief account of the history of research on pulmonary surfactant. We will then discuss the morphological aspects and composition of the pulmonary surfactant system. We describe the hydrophilic surfactant proteins A and D and the hydrophobic surfactant proteins B and C, with focus on the crucial roles of these proteins in the dynamics, metabolism, and functions of pulmonary surfactant. Next we discuss the major disorders of the surfactant system. The final part of the review will be focused on the potentials and complications of surfactant therapy in the treatment of some of these disorders. It is our belief that increased knowledge of the surfactant system and its functions will lead to a more optimal composition of the exogenous surfactants and, perhaps, widen their applicability to treatment of surfactant disorders other than neonatal respiratory distress syndrome.
MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20-40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.
Recent observations suggest that a low surface tension may be an important attribute of the lining of the air passages of the lung.1-4 The purpose of this paper is to present evidence that the material responsible for such a low surface tension is absent in the lungs of infants under 1,100-1,200 gm. and in those dying with hyaline membrane disease. The role of this deficiency in the pathogenesis of the disease is considered.
Surface tension operates so as to minimize the area of the surface. In the lungs, where the internal surface (the alveolar lining) is curved concave to the airway, the tendency of the surface to become smaller promotes collapse. Although the forces not only of surface tension but also of the elastic tissue tend to collapse the lungs, their behavior differs in one important respect. When the lung contains only a small volume of air, the elastic
Raman spectra of 2‐thiabutane have been measured in the liquid and crystalline states. Infrared spectra of these substances have been recorded in the vapor, liquid, and crystalline states. The skeletal frequencies for different rotational isomers have been calculated by the use of a potential function of the Urey‐Bradley type. It was concluded that in the gaseous and liquid states there exist both the trans and gauche isomers, of which only the former persists in the crystalline state. These two rotational isomers have been found to have nearly the same energy in the gaseous and liquid states.
Molecular structural changes due to heat-induced gelation of β-lactoglobulin were investigated by Raman scattering spectroscopy. Transparent fine-stranded gels were prepared from 15%w/v β-lactoglobulin at pH 7 or 2 by heating at 80 °C for 60 min. Opaque particulate gels were formed at pH 7 with 0.1 or 0.3 mol/dm3 NaCl or at pH 5.4. Heat-induced gelation caused a general increase in disordered secondary structural fractions and more strongly hydrogen bonded tyrosine residues. The intensity of the tryptophan vibrational band around 760 cm−1 increased on the formation of fine-stranded gels but decreased on the formation of particulate gels. A significant involvement of hydrophobic interactions on forming particulate gels was suggested by the intense band at 1345 cm−1, assigned to CH bending vibrations. Secondary structures appeared to be preserved better in particulate gels than in fine-stranded gels: a decrease in α-helix was more pronounced in fine-stranded gels, while considerable fractions of β-sheet structures remained in both types of gels, consistent with known heat-resistance of the β-barrel structure in β-lactoglobulin. Raman spectroscopy allows discrimination between gel network types at certain amino acid side chain levels as well as the secondary structure level.
Raman and infrared spectra of dialkyl disulfides were measured. Correlations of S–S and C–S stretching frequencies to molecular conformations were found for dialkyl disulfides.The molecular structure of cystine in aqueous solution was studied on the basis of the observed S–S and C–S stretching vibrations.
Incoming and reflected light waves superimpose to form a standing-wave pattern normal to a reflecting surface. For total internal reflection, a cosine distribution of the electric field amplitude in the denser medium joins onto the exponential distribution of the penetrating field in the rarer medium. The electric field amplitude at the reflecting interface is a maximum at the critical angle and decreases to zero at grazing incidence. In this paper, theoretical expressions are given for the electric field amplitudes, near the surface, which depend both on polarization and on angle of incidence. These expressions enable us to calculate from simple formulas, and without the aid of computers, the reflectivity losses resulting from the interaction of these standing waves with absorbing species, near the surface either in the rarer or denser medium. They also give us physical insight into the nature of the absorption mechanism at the reflecting interface when the reflection is frustrated. This is helpful in the fields of internal reflection optical spectroscopy and fiber optics. Experimental results, which agree with theoretical expectations, are presented. Strongest coupling is obtained by working near the critical angle for either polarization, and the absorption in the rarer medium is greater for parallel polarization than for perpendicular polarization.
The infrared and Raman spectra of several dialkyl disulfides were measured and their conformations were discussed in relation with the characteristic S–S and C–S stretching vibrations. The infrared spectra in the solid state and the temperature dependence of Raman spectra were measured for ethyl methyl disulfide and diethyl disulfide. For ethyl methyl disulfide, the gauche-gauche isomer was more stable than the trans-gauche isomer by about 0.9 kcal/mol while for diethyl disulfide the gauche-gauche-gauche isomer was more stable than the trans-gauche-gauche isomer by about 0.6 kcal/mol in the liquid state. Only the gauche-gauche form for ethyl methyl disulfide and only the gauche-gauche-gauche form for diethyl disulfide were observed in the solid state.
The molecular orientation is generally expressed by an "order parameter," which depends on both the angular position and the shape of the orientation distribution. This parameter is an average made over all orientations of the structural units studied in a sample and, consequently, a given value can correspond to different orientation distributions. In this article, model distributions are used to show the relationship between the shape, width, and angular position of the center of the orientation distribution on the coefficient, for the case where the distribution of the molecular chains exhibits cylindrical symmetry with respect to the reference direction. A significant difference is observed between the order parameters calculated for distributions of Gaussian and Lorentzian shapes with similar width at half-height. The variation of the coefficient as a function of the width at half-height, W1/2, and of the position of the center of the distribution, θC, is analyzed. Figures showing the range of W1/2–θC coordinates that can correspond to a given value are presented. As an example, the influence on the order parameter of the disorder between the different domains of phospholipid samples (mosaic spread) and of the conformational disorder in the acyl chains of these molecules is also studied. This example permits the evaluation of the magnitude of the errors that can be introduced in calculations of the tilt angle of the molecular chains in the case of distributions of finite widths or of bimodal character. Keywords: orientation, orientation function, phospholipid bilayers, conformational disorder, mosaic spread.
The α-helix, the antiparallel-chain extended conformation and the parallel-chain extended conformation of polypeptides show both parallel and perpendicular dichroism in their amide I and II infrared absorption bands. The frequencies of the amide I and II bands of these conformations (as well as those of the random coil conformation) are characteristic and now have been shown to be explicable in terms of vibrational interactions between adjacent peptide groups in the chain and through hydrogen bonds. In particular, the amide I band (1695 cm.-1) of the antiparallel-chain pleated sheet may be used in structure diagnoses of extended polypeptide chains. The amide I transition moment of the antiparallel-chain pleated sheet may be estimated from the intensity ratio of the parallel and perpendicular bands. The directions of the amide I and II transition moments of the α-helix of poly-γ-beuzyl-L-glutamate are estimated to be inclined from the helix axis by 29-34° and 75-77°, respectively. The apparent dichroic ratios of the perpendicular amide I band (1630 cm.-1) of the pleated sheet and the perpendicular amide II band (1545 cm.-1) of the α-helix indicate the degree of orientation of the fiber axes. The interpretations of the infrared spectra of several proteins have been revised.
The increasing use of infrared dichroism measurements in studying the molecular structure of fibrous proteins necessitates a critical examination of its interpretation. Relationships between transition moment direction and dichroic ratio are derived for perfectly and imperfectly oriented fibers, and the effects of reflection losses, form dichroism, and convergence are considered. Uncertainties in the assignment of the transition moment directions associated with the principal vibrations of the —CO⋅NH— group are discussed.
The Raman spectra of a series of disulfides with CS-SC dihedral angles, χ(CS-SC), in the 0 to about ±65° range (strained disulfides) have been studied in the S-S stretching region. The observed variations in the S-S stretching frequencies, νS-S), of these compounds, all of which contain the CαCβSSCβCα unit, are attributed to differences in the degree of substitution at their β carbons and to differences in conformation about their S-S bonds. When the effects of substitution at the β carbons were taken into account, the effects leading to reduced values of χ(CS-SC) were isolated. As χ(CS-SC) is lowered from near 90° to near 0°, an approximately linear reduction in ν(S-S) is observed. These experimental findings are in good qualitative agreement with the trend in ν(S-S) reported previously, and also calculated previously for the model compound dimethyl disulfide using the CNDO/2 molecular orbital method. The use of values of ν(S-S) for the estimation of χ(CS-SC) for cystine residues in proteins and other primary disulfides is discussed.
The Raman spectra of a series of disulfides structurally related to cystine have been studied in the 250-650-cm-1 region. These compounds all contain the unstrained CCSSCC structural unit and have very similar conformations about their S-S bonds (i.e., CS-SC dihedral angles near ±90°), but a wide variety of conformations about their C-S bonds. The effects of varying conformation about their C-S bonds on their S-S stretching frequencies, ν(S-S), have been examined. From the spectra of those crystalline disulfides whose structures are known from x-ray studies, it has been found that primary disulfides with trans and either of two nonequivalent gauche conformations about their C-S bonds (SS-CC dihedral angles of roughly 180 and ±60°, respectively) all have values of ν(S-S) of about 510 cm-1. This is in disagreement with the frequency-conformation correlation proposed previously by other workers, whereby it was thought that CCSSCC moieties with trans conformations about their C-S bonds have values of ν(S-S) of about 540 cm-1. Instead, it is suggested that a value of ν(S-S) of 540 cm-1 in primary disulfides arises from the presence of conformations (referred to as A conformations) with small (∼30°) SS-CC dihedral angles. The existence of these A conformations has been suggested by recent CNDO/2 calculations carried out on several alkyl disulfides. These A conformations are shown to comprise ∼20% of all occurrences of the values of χ(SS-CC) found for cystine residues in eight proteins, as determined by x-ray crystallography. The use of the values of ν(S-S) for the determination of conformation about the C-S bonds of cystine residues in proteins is discussed.
In conjunction with a gas-phase electron diffraction investigation of the structures of dimethyl and methyl ethyl disulfides (accompanying paper), the Raman spectra of dimethyl, methyl ethyl, and diethyl disulfides have been studied from 80 to 800 cm-1. The variations in the positions and intensities of the fundamentals in the spectra of methyl ethyl and diethyl disulfides as a function of temperature from ∼77 to 320 K have been found to be consistent with the presence of three rotational isomers differing in their conformations about their C-S bonds. This disagrees with the results of an earlier study of these same molecules which were interpreted in terms of the existence of only two rotamers. Bands due to the high energy rotamer, whose S-S stretching frequency lies at 524 cm-1, nearly disappear when the temperature is reduced to ∼77 K. However, significant amounts of the other two rotamers, both of which have S-S stretching frequencies of 508 cm-1, persist down to ∼77 K, indicating that they are of nearly equal energy. The results of the electron diffraction study on methyl ethyl disulfide indicate the existence of a substantial fraction of molecules with a trans conformation about their C-S bonds. In addition, Raman data on dithioglycolic acid, which is known from x-ray studies to have a trans conformation about its C-S bonds, indicate that its S-S stretching frequency is about 508 cm-1. These two results imply that the rotamer responsible for the S-S stretching mode at 524 cm-1 in the alkyl disulfides studied here is not the trans rotamer, as has been assumed by other workers.
In the present study, Fourier transform infrared-attenuated total reflection spectroscopy has been used to characterize Langmuir-Blodgett films of purple membranes deposited on Ge crystals at different surface pressures. The results obtained indicate that the average angle of the protein's α-helices with respect to the interface normal remains unchanged with increasing lateral pressure. Moreover, the total absorbance of the films as a function of molecular area suggests that a multilayer structure is formed between 40 and 46 mN m−1.
MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20–40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.
Laser-Raman spectra of native lysozyme in aqueous solution (7%, pH 5, 0.1 M NaCl) have been observed in the temperature range 32–76°C. In this range there is very little quantitative change in the spectra, from which the conclusion is drawn that any conformational change in the native structure is confined mainly to the sidechains. The disulfide bonds are intact at 76°C. If the protein solution is heated for 2 h at 100°C, the resultant irreversible denaturation produces a gel whose Raman spectrum at 32°C shows differences from that of the native protein, particularly in the amide I, amide III and disulfide regions. These are such as to imply a disordering of the polypeptide backbone, including changes in its conformation near the disulfide cross-links. At most, only one of the four disulfide bonds disappears with this treatment, though the data are consistent with no loss of bonds, but with a substantial alteration in their geometries.
Surfactant protein B is a small homodimeric protein that is found tightly associated with surfactant lipids in the alveolar space. In this review, we discuss the actions of SP-B on phospholipid membranes using information predominantly obtained from model membrane systems. We try to correlate these model actions with current concepts of SP-B structure and proposed biological functions. These functions may include critical roles in the intracellular assembly of surfactant through a role in lamellar body organogenesis, the structural rearrangement of secreted surfactant lipids into tubular myelin, and the subsequent rapid insertion of secreted surfactant phospholipids into the surface film itself. The relevance of SP-B to human biology is emphasized by the fatal respiratory distress that is associated with a genetic deficiency of SP-B and the important role of SP-B in certain exogenous surfactant formulations in wide clinical use.
Pressure-area isotherms, Brewster angle microscopy, and grazing incidence X-ray diffraction measurements reveal that human lung surfactant protein SP-B1-78 and the dimer of the amino terminus dSP-B1-25 modify the phase behavior of lipid mixtures consisting of dipalmitoylphosphatidylcholine/palmitoyl-oleyl-phosphatidylglycerol/palmitic acid (DPPC/POPG/PA). The addition of SP-B increases the fraction of fluid phase in the liquid-expanded/liquid-condensed two-phase region. Brewster angle microscopy enabled the visualization of a fluid network, which separates the condensed phase domains. This network is stabilized by SP-B adsorption. GIXD measurements show that SP-B also alters the structure of the condensed chain lattice leading to higher tilt and increased area per hydrocarbon chain. The comparison of SP-B1-78 with the shorter peptide dSP-B1-25 exhibits, that the dimer alters the lipid order more drastically. The larger effects found for dSP-B1-25 were explained using a model that assumes a partial incorporation of the peptide into the layer. The specific behavior of the dimer could enhance the activity of the peptide as found in recent animal model studies. This is the first investigation showing a systematic influence of SP-B on the condensed chain lattice of phospholipids, thus verifying that SP-B not only interacts with the expanded phase, but also interactions with the condensed phase lipids have to be taken into account which might be essential for proper peptide function.
The doublet at 850 and 830 cm-1 in the Raman spectra of proteins containing tyrosyl residues has been examined as to its origin and the relation of its components to the environment of the phenyl ring, the state of the phenolic hydroxyl group, and the conformation of the amino acid backbone. Raman spectral studies on numerous model molecules related to tyrosine, including certain deuterium derivatives, show that the doublet is due to Fermi resonance between the ring-breathing vibration and the overtone of an out-of-plane ring-bending vibration of the para-substituted benzenes. Further examination of the effects of pH and solvents on the Fermi doublet and of the crystallographic data demonstrates that the intensity ratio of the two components depends on changes in the relative frequencies of the two vibrations. These in turn are found to be sensitive to the nature of the hydrogen bonding of the phenolic hydroxyl group of its ionization, but much less so to the environment of the phenyl ring and the conformation of the amino acid backbone. By use of the relative intensities of the doublet in model systems where the phenolic hydroxyl group is strongly hydrogen-bonded, weakly hydrogen-bonded, free or ionized, the reported Raman intensities of the doublets observed in the Raman spectra of several proteins have been interpreted. The results are compared with those obtained by other techniques.
Polarized Fourier transform infrared spectroscopy has been used to study the structure of purple membrane from Halobacterium halobium. Membranes were oriented by drying a suspension of membrane fragments onto Irtran-4 slides. Dichroism measurements of the amide I, II and A peaks were used to find the average spatial orientation of the bacteriorhodopsin alpha-helices. By deriving a function that relates the observed dichroism to the orientational order parameters for the peptide groups, helical axis distribution, and mosaic spread of the membranes, the average orientation of the alpha-helices was found to lie in a range of less than 26 degrees away from the membrane normal, agreeing with electron microscopic measurements. The frequency of the amide I and A peaks is at least 10 cm-1 higher than values found for most alpha-helical polypeptides and proteins. This may indicate that bacteriorhodopsin contains distorted alpha-helical conformations.
Laser Raman spectra of neurotoxins of Pelamis platurus (yellow-bellied sea snake) and Laticauda semifasciata (broad-banded blue sea snake) were investigated. The amide I band appeared at 1672 cm-1for both toxins, which presents an indication of anti-parallel β structure. Since this agrees well with the result from the CD-ORD studies of snake neurotoxin, it was concluded that snake neurotoxins mainly consist of β structure. The amide III band appeared at 1245 cm-1for P. platurus toxin and 1248 cm-1for L. semifasciata toxin.
The four disulfide bonds present in the toxin have a very similar geometry. After vigorous heat treatment, the backbone configuration of the toxin molecule basically remained the same although it was partially denatured. The major peak at 512 cm-1was not altered by the heat treatment but a new shoulder appeared at 546 cm-1. This suggests that a new type of S-S stretching vibration (trans-gauche-trans) was produced as a result of heat treatment. However, the majority of the S-S vibrations remained in the gauche-gauche-gauche orientation. A substantial change in the interactions between a tyrosine aromatic ring and neighboring residues was apparently the alteration caused by the heat treatment.
The disulfide bridges in porcine hydrophobic surfactant protein B (SP-B) were determined. Results show that three intrachain bridges link half-cystine residues 8 and 77, 11 and 71, and 35 and 46, respectively. This gives SP-B an appearance of three loops, a central big loop surrounded by two smaller ones. In the major form of SP-B, the remaining half-cystine, Cys-48, is probably interchain-linked to its counterpart in another molecule, compatible with the existence of dimeric molecules. A minor fraction, with monomeric SP-B but also lacking free thiols, could be due to polypeptides having Cys-57 (instead of Leu in the major form) and hence an additional intrachain bond (Cys-48-Cys-57). Notably, one of the three intrachain bonds common to all SP-B molecules is analogous to one of the disulfide linkages in the kringle structure of complex serine proteases. SP-B and kringles are also similar in size and in positions of half-cystine residues. SP-B and the kringle of coagulation factor XII exhibit 26% residue identity. This structural similarity of SP-B to a binding domain could reflect functional homology, compatible with the notion that SP-B interacts with surfactant anionic phospholipids, which is also in agreement with an SP-B excess of basic residues. Finally, weak similarities between the perform of SP-B and complex serine proteases are also found. This has implications on further possible relationships between kringles, serine proteases, and antiproteases.
Excellent results have been obtained for the Fmoc solid-phase syntheses of peptides using the activating reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU). Activation occurs very rapidly in N,N-dimethylformamide and N-methyl-pyrrolidone, optimal solvents for peptide-resin solvation. It has been observed that complete coupling reactions occur in only 10-30 min. Residues such as Arg, Ile, Leu and Val, which often require double coupling by other activation methods, react with high efficiency by single coupling when HBTU is used. The Fmoc/HBTU chemistry has recently been applied to the peptide synthesizers. The incorporation of trityl side-chain protection for Fmoc-Asn and Fmoc-Gln further enhances coupling efficiencies in difficult sequences.
SP-B is a protein in pulmonary surfactant that is, in greatest part, responsible for resistance to surface tension and prevention
of collapse of pulmonary alveoli. Peptides of 21 residues, synthesized following the sequence of SP-B or resembling the hydrophobic
and hydrophilic domains of SP-B (such as RLLLLRLLLLRLLLLRLLLLR, R, Arg, and L, Leu), enhanced the abilities of phospholipids
to reduce surface tension both in vitro and in vivo. Intermittent positively charged residues were essential for this activity.
The SP-B-like peptides were found by tryptophan fluorescence to partition within the phospholipid layer in contact with both
polar head groups and acyl side chains. These data, together with findings that the SP-B-related peptides increase inter-
and intramolecular order of the phospholipid layer, suggest that SP-B resists surface tension by increasing lateral stability
of the phospholipid layer.
Pulmonary surfactant contains at least three unique proteins: SP-A, SP-B and SP-C. SP-B and SP-C from bovine surfactant are markedly hydrophobic and have molecular masses between 3 and 26 kDa. We identify surfactant proteins under nonreducing conditions on polyacrylamide gels with approximate molecular mass of 5, 14, 26 kDa (SP-5, 14, 26) when organic solvent-soluble material is eluted from a Sephadex LH-20 size exclusion column followed by separation on a high-performance reverse-phase chromatography system. These bands correspond to monomeric SP-C, oligomeric SP-C and oligomeric SP-B, respectively. Computer analysis (Eisenberg-hydrophobic moment) of sequences for these proteins suggests that SP-B contains surface-seeking amphiphilic segments. In contrast, SP-C resembles a more hydrophobic transmembrane anchoring peptide. Dispersions containing dipalmitoylphosphatidylcholine, phosphatidylglycerol, palmitic acid and multimeric SP-B and SP-C duplicate the surface activity of natural surfactant when assayed in a pulsating bubble surfactometer. We speculate that oligomers of SP-B and monomers and oligomers of SP-C may act cooperatively in affecting surfactant function. An important function of SP-B and SP-C may be to affect the ordering of surfactant lipids so that rates of transport of surfactant lipids to the hypophase surface in the alveoli are enhanced.
The effects of bovine pulmonary surfactant-associated protein B (SP-B) on molecular packing of model membrane lipids (7:1 DPPC/DPPG) were studied by fluorescence anisotropy. The bilayer surface was markedly ordered by SP-B below the gel to fluid phase transition temperature (Tc) while it was only slightly ordered above this temperature as indicated by surface-sensitive probes 6-NBD-PC and 6-NBD-PG. The effects of SP-B on fluorescence anisotropy were concentration dependent, reaching maximal activity at 1-2% protein to phospholipid by weight. Anisotropy measurements of interior-selective fluorescent probes (cis-parinaric acid and DPH) imply that addition of SP-B into the phospholipid shifted the Tc of the model membrane but did not alter lipid order at the membrane interior. Since fluorescence anisotropy studies with trans-parinaric acid, an interior-sensitive probe with high affinity for gel-phase lipids, did not detect any changes in lipid packing or Tc, it is likely that SP-B resides primarily in fluid-phase domains. Fluorescence lifetime measurements indicated that two conformers of the NBD-PC probe exist in this DPPC/DPPG model membrane system. Fluorescence intensity measurements generated with NBD-PC and NBD-PG, in conjunction with information from lifetime measurements, support the concept that SP-B increases the distribution of the short-lifetime conformer in the gel phase. In addition, the anisotropy and intensity profiles of NBD-PG in the model membrane indicate that bovine SP-B interacts selectively with phosphatidylglycerol.
We present a prospective study, designed to evaluate surfactant abnormalities in severely injured patients during the course of post-traumatic pulmonary dysfunction. Serially obtained bronchoalveolar lavage fluids from noncontused lung areas (in total, 132 samples from 17 patients) were analyzed for alveolar phospholipid composition and surfactant function in vitro during the first 14 days after trauma. The data were compared with those of 29 lavage samples obtained from 10 healthy control subjects and correlated to severity of respiratory failure. In the traumatized patients, the total lavage phospholipid content was unchanged, but there was a progressive decrease in the relative amounts of phosphatidylcholine (%PC) and phosphatidylglycerol and an increase in phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin. These alterations were paralleled by a marked decrease in the hysteresis area of the surface tension isotherm. The decrease in %PC and reduction of hysteresis area were significantly correlated. The alterations in alveolar phospholipid composition and in vitro surfactant function were more pronounced in patients with severe respiratory failure. There was a significant inverse correlation between severity of respiratory dysfunction and %PC or hysteresis area for all traumatized patients. Protein leakage into the alveolar space was significantly higher in patients with severe respiratory failure and appeared to precede surfactant abnormalities in such patients. The neutrophil content in the alveolar space was markedly increased in all patients with multiple injuries however, no significant correlation with the noted alterations in alveolar phospholipid composition or surfactant function was found. We concluded that surfactant abnormalities occur during the course of post-traumatic pulmonary dysfunction and are correlated with the severity of respiratory failure.
The low-molecular-mass surfactant protein fraction, soluble in chloroform/methanol, contains at least two separate polypeptide chains. The 8-kDa form (type I) was isolated, [14C]carboxymethylated after reduction, and submitted to structural analysis. Its highly hydrophobic nature complicated purification, proteolytic cleavages, and sequence analysis. Acid hydrolysis in 6 M HCl for 7 days was necessary for release of branched-chain residues in full yield. Pepsin was the only enzyme found to cleave the surfactant protein and was used to complement peptide generation by chemical cleavage with CNBr. The primary structure deduced consists of 79 residues with 8 half-cystine residues, and a total of 39% branched-chain hydrophobic residues. However, 11 residues are charged at physiological pH, and all properties of the primary structure are not entirely outstanding in relation to those of other proteins. Hydrophobic segments, coupled with a presumably tight folding from the presence of disulfide bridges, probably explain the unusual properties and the solubility in organic solvents.
Possible conformational changes of α-lactalbumin associated with lyophilization and crystallization were investigated in detail by Raman scattering technique It was found that lyophilization altered the protein three-dimensional structure considerably, but crystallization produced no detectable effect on backbone conformation. This conclusion is the same as that reached earlier for lysozyme. In the amide III region (both crystals and solution spectra) three well-defined peaks were observed at 1274, 1260, and 1238 cm-1 very similar to those of lysozyme at 1272, 1258, and 1238 cm-1, respectively, indicating that α-lactalbumin may have a conformation similar to that of hen's egg-white lysozyme. This is consistent with the "analogy" molecular model proposed by Browne, et al. To demonstrate the sensitivity of the amide III contour with respect to change in α-lactalbumin backbone conformation, Raman spectra of aqueous solution at pH 6.6 and 2.1 were compared. Spectral evidence was presented that the distinct feature of an extremely sharp peak at 1361 cm-1 in both α-lactalbumin and lysozyme was related to some specific interactions involving those "buried" indole rings.
Laser-excited Raman spectra of a simple native protein, lysozyme, in aqueous solution are reported and partially interpreted with the help of the spectra of its constituent amino acids. Raman spectroscopy should be useful in providing direct evidence concerning the presence and number of disulfide cross-links in proteins, and may also be useful in studying the local conformation of the CSSC group. The aromatic side-groups of phenylalanine, tryptophan and tyrosine give rise to very intense and sharp lines. These lines are not sensitive to changes in conformation or state of aggregation. The peptide CONH group gives rise to two characteristic lines near 1660 (amide I) and 1260 cm−1 (amide III). They appear to be potentially useful in assessment of conformational changes caused by denaturation. Lines in the region from 800 to 1150 cm−1 arising from CC and CN stretching vibrations are also expected to be conformation-dependent.
Laser-excited Raman spectra of native ribonuclease and α-chymotrypsin and of the polypeptide poly-l-glutamic acid have been determined in aqueous solution. With the help of previously determined quantitative spectra of the amino acids, a considerable portion of the spectral details has been interpreted. The strong Raman lines due to the aromatic side chains of the amino-acid residues are clearly observed and are not affected by conformations of the proteins. The bond-stretching vibration of the disulfide link shows up strongly in the spectrum of ribonuclease at a measurably different position (516 cm−1) from its location in lysozyme (509 cm−1). The intensity ratio of the C-S to S-S lines is an order of magnitude larger than in lysozyme. Both of these observations suggest that the conformations of the C-S-S-C cross-links in the two proteins are significantly different. The spectrum of poly-l-glutamic acid at pH 10 was observed as a possible model for the effects of denaturation. While the spectra of the denatured proteins have not yet been obtained, comparison of the native spectra with that of poly-l-glutamic acid shows for the latter the expected broadening and overlapping of peaks in the amide I and amide III regions which are sharper and resolved into several components in the protein spectra.
Application of vibrational spectroscopy to the problem of structure determination of molecules of biological interest goes back to the early uses of raman and infrared spectroscopy in the study of organic molecules. For reviews of earlier work the reader is referred to compilations by Kohlrausch (1943) and by Jones and Sandorfy (1956), whereas more recently a comprehensive discussion has been presented by Bellamy (1975). These compilations accentuate the correlation of vibrational spectra with molecular structure from an essentially empirical point of view and culminate in the establishment of empirical correlation charts. For typical examples the reader is referred to Weast (1974) and Bellamy (1975). There have been many treatments of the theoretical basis of molecular vibrational spectroscopy. Among them the classical work by Herzberg (1945) and by Wilson et al. (1955) should be mentioned. Applications of infrared spectroscopy (IR) to structure problems of biological interest have been summarized by Susi (1969), Fraser and MacRae (1973), and Wallach and Winzler (1974). It was remarked quite eraly that relevant structural information about biological systems often requires study in aqueous solution, which forms the natural environment for most biologically important systems. Besides critical control of experimental conditions and samples the conventional methods of raman spectroscopy may be applied to aqueous solutions in a quite straightforward manner, cf. the contribution by Lord and Mendelson, Chapter 8.
The tumour-lysing and antimicrobial polypeptide NK-lysin and the pulmonary surfactant-associated polypeptide SP-B exhibit 24% residue identities (49% similarities), including six half-cystine residues in the same disulphide bonding pattern, and similar far-UV circular dichroism spectra corresponding to 45-55% alpha-helix and 20-25% beta-sheet structures. From this, we conclude that the conformations of NK-lysin and SP-B are similar. In contrast, the functional properties of the two proteins are dissimilar: SP-B does not exhibit antibacterial activity and NK-lysin does not significantly effect phospholipid spreading at an air/water interface. Saposins, which solubilize lipids and activate lysosomal hydrolases, the pore-forming amoebapores, and parts of acid sphingomyelinase and acyloxyacylhydrolase, also share 18-27% sequence identities with NK-lysin (and SP-B), including the six conserved half-cystine residues. The inclusion of NK-lysin extends the family of saposin-like polypeptides, all members of which appear to interact with lipids. Strictly conserved structural features with implications for helix topology and lipid interactions are observed.
The structural and functional integrity of pulmonary surfactant depends on several specific proteins. Two of these, SP-A and SP-D, are large and water-soluble, while SP-B and SP-C are small and very hydrophobic. SP-A is an 18-mer of 26 kDa polypeptide chains and contains N-linked oligosaccharides. Structurally, it can be characterized as a collagen/lectin hybrid. Together with SP-B, SP-A is required for conversion of secreted endogenous surfactant to tubular myelin in the alveolar lining. It also regulates surfactant secretion and reuptake of surfactant lipids by type II cells; these functions are probably receptor mediated. SP-D, a 12-mer of 39 kDa polypeptide chains, is a collagenous glycoprotein with structural similarities to C-type lectins. Both SP-A and SP-D stimulate alveolar macrophages. SP-B is a 79-residue polypeptide that contains three intrachain disulphide bridges. It exists mainly as a homodimer, which is strongly positively charged and may selectively remove anionic and unsaturated lipid species from the alveolar surface film, thereby increasing surface pressure. SP-C is a mainly alpha-helical, extraordinarily hydrophobic polypeptide containing 35 amino acid residues and covalently linked palmitoyl groups. Its alpha-helical portion is inserted into surfactant lipid bilayers. SP-C accelerates the adsorption of lipid bilayers to an interfacial monolayer. In babies with respiratory distress syndrome, the clinical response to treatment with surfactant containing SP-B and SP-C is much faster than in babies treated with protein-free synthetic surfactant. We speculate that, in the near future, surfactant preparations based on recombinant hydrophobic proteins will be available for clinical use.
To determine the molecular defect accounting for the deficiency of pulmonary surfactant protein B (SP-B) in full-term neonates who died from respiratory failure associated with alveolar proteinosis, the sequence of the SP-B transcript in affected infants was ascertained. A frameshift mutation consisting of a substitution of GAA for C in codon 121 of the SP-B cDNA was identified. The three affected infants in the index family were homozygous for this mutation, which segregated in a fashion consistent with autosomal recessive inheritance of disease. The same mutation was found in two other unrelated infants who died from alveolar proteinosis, one of whom was also homozygous, and in the parents of an additional unrelated, affected infant, but was not observed in 50 control subjects. We conclude that this mutation is responsible for SP-B deficiency and neonatal alveolar proteinosis in multiple families and speculate that the disorder is more common than was recognized previously.
ARDS includes a complex series of events leading to alveolar damage, high permeability pulmonary edema, and respiratory failure. The endogenous pulmonary surfactant system is crucial to maintaining normal lung function, and only recently has it been appreciated that alterations in the surfactant system significantly contributed to the pathophysiology of the lung injury of patients with ARDS. Through a combination of analyzing BAL samples from patients with ARDS and extensive animal studies, there have been significant insights into the variety of surfactant abnormalities that can occur in injured lungs. These include altered surfactant composition and pool sizes, abnormal surfactant metabolism, and inactivation of alveolar surfactant by serum proteins present within the airspace. Positive effects of exogenous surfactant administration on acute lung injury have been reported. There is now a prospective, randomized clinical trial evaluating the efficacy of aerosolized exogenous surfactant in patients with ARDS. This trial has demonstrated improvements in gas exchange and a trend toward decreased mortality in response to the surfactant. Despite these encouraging results, there are multiple factors requiring further investigation in the development of optimal surfactant treatment strategies for patients with ARDS. Such factors include the development of optimal surfactant delivery techniques, determining the ideal time for surfactant administration during the course of injury, and the development of optimal exogenous surfactant preparations that will be used to treat these patients. With further clinical trials and continued research efforts, exogenous surfactant administration should play a useful role in the future therapeutic approach to patients with ARDS.
Congenital pulmonary alveolar proteinosis is an uncommon cause of respiratory failure in full-term newborns1–4. Although its histopathological appearance is similar to that of the alveolar proteinosis observed in older children and adults,5 the congenital form of the illness follows a different clinical course. All reported infants with congenital alveolar proteinosis have died within the first year of life despite maximal medical therapy. The incidence and cause of congenital alveolar proteinosis are unknown. Familial cases have been reported, and it has been speculated that the cause is an inborn error of surfactant metabolism4. In this report we describe . . .
Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids.
The Silences of the Archives, the Reknown of the Story.
The Martin Guerre affair has been told many times since Jean de Coras and Guillaume Lesueur published their stories in 1561. It is in many ways a perfect intrigue with uncanny resemblance, persuasive deception and a surprizing end when the two Martin stood face to face, memory to memory, before captivated judges and a guilty feeling Bertrande de Rols. The historian wanted to go beyond the known story in order to discover the world of the heroes. This research led to disappointments and surprizes as documents were discovered concerning the environment of Artigat’s inhabitants and bearing directly on the main characters thanks to notarial contracts. Along the way, study of the works of Coras and Lesueur took a new direction. Coming back to the affair a quarter century later did not result in finding new documents (some are perhaps still buried in Spanish archives), but by going back over her tracks, the historian could only be struck by the silences of the archives that refuse to reveal their secrets and, at the same time, by the possible openings they suggest, by the intuition that almost invisible threads link here and there characters and events.
NK-lysin is the first representative of a family of sequence related proteins--saposins, surfactant-associated protein B, pore forming amoeba proteins, and domains of acid sphingomyelinase, acyloxyacylhydrolase and plant aspartic proteinases--for which a structure has been determined.