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Investigating the Binding Mechanism of Temporin Rb with Human Serum Albumin, Holo Transferrin, and Hemoglobin using Spectroscopic and Molecular Dynamics Techniques
Molecular dynamics (MD) simulation was used to study the influence of electric field on Glucagon-like Peptide-2 (GLP-2). Different electric field strengths (0 V/nm ≤ E ≤ 1 V/nm) were mainly carried out on GLP-2. The structural changes in GLP-2 were analyzed by the Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (Rg), Solvent Accessible Surface Area (SASA), Secondary Structure and the number of hydrogen bonds. The stable α—helix structure of GLP-2 was unwound and transformed into an unstable Turn and Coil structure since the stability of the GLP-2 protein structure was reduced under the electric field. Our results show that the degree of unwinding of the GLP-2 structure was not linearly related to the electric field intensity. E = 0.5 V/nm was a special point where the degree of unwinding of the GLP-2 structure reached the maximum at this electric field strength. Under a weak electric field, E < 0.5 V/nm, the secondary structure of GLP-2 becomes loose, and the entropy of the chain increases. When E reaches a certain value (E > 0.5 V/nm), the electric force of the charged residues reaches equilibrium, along the z-direction. Considering the confinement of moving along another direction, the residue is less free. Thus, entropy decreases and enthalpy increases, which enhance the interaction of adjacent residues. It is of benefit to recover hydrogen bonds in the middle region of the protein. These investigations, about the effect of an electric field on the structure of GLP-2, can provide some theoretical basis for the biological function of GLP-2 in vivo.
The fluorescence quenching by protons is a universal phenomenon but the mechanism remains unclear. Here, we take the fluorescent amide-terminated carbon dots as a prototype to study the proton fluorescence quenching mechanism by using both experiments and timedependent density functional theory calculations. The study reveals that when an approached proton is captured by the weakly negatively charged fluorophore group of the colloidal carbon dot, it will substantially change the electron wavefunctions owing to the strong proton-electron interaction, and this leads to highly diminished energy gap and resultant fluorescence quenching in the visible spectral region. The protons generated by hydrolysis of various types of metal ions also exhibit fruitful fluorescence quenching and the quenching efficiency is roughly proportional to the hydrolysis constant of the metal ion. This fluorescence quenching mechanism is quite distinct from the conventional ones involving electron or energy transfer.
The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 104 M−1 has been assessed through a Stern–Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.
Hemoglobin is one of the most widely studied proteins genetically, biochemically, and structurally. It is an oxygen carrying tetrameric protein that imparts the characteristic red color to blood. Each chain of hemoglobin harbors a heme group embedded in a hydrophobic pocket. Several studies have investigated structural variations present in mammalian hemoglobin and their functional implications. However, camel hemoglobin has not been thoroughly explored, especially from a structural perspective. Importantly, very little is known about how the heme group interacts with hemoglobin under varying conditions of osmolarity and temperature. Several experimental studies have indicated that the tense (T) state is more stable than the relaxed (R) state of hemoglobin under normal physiological conditions. Despite the fact that R state is less stable than the T state, no extensive structural dynamics studies have been performed to investigate global quaternary transitions of R state hemoglobin under normal physiological conditions. To evaluate this, several 500 ns all-atom molecular dynamics simulations were performed to get a deeper understanding of how camel hemoglobin behaves under stress, which it is normally exposed to, when compared to human hemoglobin. Notably, camel hemoglobin was more stable under physiological stress when compared to human hemoglobin. Additionally, when compared to camel hemoglobin, cofactor-binding regions of hemoglobin also exhibited more fluctuations in human hemoglobin under the conditions studied. Several differences were observed between the residues of camel and human hemoglobin that interacted with heme. Importantly, distal residues His58 of α hemoglobin and His63 of β hemoglobin formed more sustained interactions, especially at higher temperatures, in camel hemoglobin. These residues are important for oxygen binding to hemoglobin. Thus, this work provides insights into how camel and human hemoglobin differ in their interactions under stress.
Streptococcus pyogenes is a significant pathogen that causes infections of the skin and the upper respiratory tract. Cross-resistance and multi-drug resistance have emerged in S. Py-ogenes strains, making them more dangerous and pathogenic. Hence, it has become critical to identify and develop therapeutic methods that present novel avenues for S. Pyogenes in-fections. In the current study, a subtractive proteomics approach has been employed to core proteome of four strains of S. pyogenes using several bio-informatics softwares and servers. Core proteome consists of 1324 proteins and 302 essential proteins were predicted from them. These essential proteins were analyzed using BLASTp against human proteome and the number of potential targets was reduced to 145. In a subcellular localization study, 46 proteins with cytoplasmic localization were predicted for metabolic pathway analysis. Only two cytoplasmic proteins i-e Chromosomal replication initiator protein DnaA, and Two-component response regulator (TCR) were discovered to have the potential to be novel drug target candidates. 3D structure prediction of target proteins was carried out via the Swiss Model server. Molecular docking approach was employed to screen the 1000 phyto-chemicals library against the interacting residues of target proteins through MOE software. Further, the docking studies were validated by running molecular dynamics simulation and highly popular binding free energy appraoches of MM-GBSA and MM-PBSA. The findings revealed a promising candidate as a novel target against S. pyogenes infections
The data provided and described here give insight into the solution dynamics of the dimer of human EpCAM ectodomain (EpEX). As the starting point, crystal structure of EpEX non-covalent dimer was used (PDB ID 4MZV). The coordinates of solvent-embedded dimer were used to generate a topology file, which was in turn used for all-atom molecular dynamics (MD) simulation run of 20 ns length using full-system periodic electrostatics at a constant temperature of 310 K and a constant pressure of 1 atm. The MD trajectory file (part of this dataset) contains 4000 frames corresponding to recording/sampling atom positions every 5 ps. The simulation run was then analyzed in terms of root mean square deviations (RMSD) of protein atoms, and non-covalent inter-subunit interactions. The MD trajectory and analyzed data enable—in contrast to the static crystal structure—detailed analysis of solution-like protein structural dynamics and support design of EpCAM-targetting binders and structure-based analysis of EpCAM interactome.
The molecular interactions of anionic tetrasulfonate phenyl porphyrin (TPPS) with poly(amido amine) (PAMAM) dendrimers of generation 2.0 and 4.0 (G2 and G4, respectively) forming H- or J-aggregates, as well as with human and bovine serum albumin proteins (HSA and BSA), were reviewed in the context of self-assembly molecular complementarity. The spectroscopic studies were extended to the association of aluminum phthtalocyanine (AlPCS4) detected with a PAMAM G4 dendrimer with fluorescence studies in both steady state and dynamic state, as well as due to the fluorescence quenching associated to electron-transfer with a distribution of lifetimes. The functionalization of TPPS with peripheral substituents enables the assignment of spontaneous pH-induced aggregates with different and well-defined morphologies. Other work reported in the literature, in particular with soft self-assembly materials, fall in the same area with particular interest for the environment. The microencapsulation of TPPS studies into polyelectrolyte capsules was developed quite recently and aroused much interest, which is well supported and complemented by the extensive data reported on the Imaging Microscopy section of the Luminescence of Porphyrins and Phthalocyanines included in the present review.
The scientific community is continuously working to discover drug candidates against potential targets of SARS-CoV-2, but effective treatment has not been discovered yet. The virus enters the host cell through molecular interaction with its enzymatic receptors i.e., ACE2 and TMPRSS2, which, if, synergistically blocked can lead to the development of novel drug candidates. In this study, 1503 natural bioactive compounds were screened by HTVS, followed by SP and XP docking using Schrodinger Maestro software. Bio-0357 (protozide) and Bio-597 (chrysin) were selected for dynamics simulation based on synergistic binding affinity on S1 (docking score -9.642 and -8.78 kcal/mol) and S2 domains (-5.83 and -5.3 kcal/mol), and the RMSD, RMSF and Rg analyses showed stable interaction. The DFT analysis showed that the adsorption of protozide/chrysin, the band gap of protozide/chrysin-F/G reduced significantly. From SERS, results, it can be concluded that QDs nanocluster will act as a sensor for the detection of drugs. The docking study showed Bio-0357 and Bio-0597 bind to both S1 and S2 domains through stable molecular interactions, which can lead to the discovery of new drug candidates to prevent the entry of SARS-CoV-2. This in-silico study may be helpful to researchers for further in vitro experimental validation and development of new therapy for COVID-19.
Antimicrobial peptides, as an important member of the innate immune system, have various biological activities in addition to antimicrobial activity. There are some AMPs with antidiabetic activity, especially those isolated from amphibians. These peptides can induce insulin release via different mechanisms based on peptide type. In this review study, we collected all reported AMPs with antidiabetic activity. We also analyze the sequence and structure of these peptides for evaluation of sequence and structure effect on their antidiabetic activity. Based on this review, the biggest peptide family with antidiabetic activity is temporins with nine antidiabetic peptides. Frogs are the most abundant source of antidiabetic peptides. Bioinformatics analysis showed that an increase of positive net charge and a decrease of hydrophobicity can improve the insulinotropic effect of peptides. Peptides with higher positive net charge and Boman index showed higher activity. Based on this review article, AMPs with antidiabetic activity, especially those isolated from amphibians, can be used as novel antidiabetic drug for type 2 diabetes disease. So, amphibians are potential sources for active peptides which merit further evaluation as novel insulin secretagogues. However, strategy for the increase of stability and positive activity as well as the decrease of negative side effects must be considered.
The interaction between human serum transferrin (hTf) and three promising organometallic Ru (II)- (η⁵-C5H5) derived complexes, that have already shown strong in vitro cytotoxicity towards human cancer cell lines, has been investigated using fluorescence spectroscopic techniques. The results suggested that the formation of Ru-hTf systems involves a dynamic collision. The binding process occurs spontaneously (ΔG < 0), mainly driven by hydrophobic interactions. Additional docking studies show that all complexes bind preferably to a specific hydrophobic pocket in the C2-subdomain as already observed for other metal-cyclopentadienyl (MCp) complexes and are in agreement with the experimental results. With these studies we hope to contribute to the understanding of the mechanism of action of these promising cytotoxic agents, thus providing clues for a more rational design.
Graphic abstract
Protein therapeutics carry inherent limitations of membrane impermeability and structural instability, despite their predominant role in the modern pharmaceutical market. Effective formulations are needed to overcome physiological and physicochemical barriers, respectively, for improving bioavailability and stability. Knowledge of membrane affinity, cellular internalization, encapsulation, and release of drug-loaded carrier vehicles uncover the structural basis for designing and optimizing biopharmaceuticals with enhanced delivery efficiency and therapeutic efficacy. Understanding stabilizing and destabilizing interactions between protein drugs and formulation excipients provide fundamental mechanisms for ensuring the stability and quality of biological products. This article reviews the molecular studies of biologics using solution and solid-state NMR spectroscopy on structural attributes pivotal to drug delivery and stability. In-depth investigation of the structure-function relationship of drug delivery systems based on cell-penetrating peptides, lipid nanoparticles and polymeric colloidal, and biophysical and biochemical stability of peptide, protein, monoclonal antibody, and vaccine, as the integrative efforts on drug product design, will be elaborated.
UV-visible spectroscopy and synchronous fluorescence spectroscopy (SFS) combined with two-dimensional correlation spectroscopy (2D-COS) were applied for extracting fluorescence components, tracing organic functional groups, and revealing variations of dissolved organic matter (DOM) in Puhe River. Water samples were collected from the mainstream and two tributaries (Nanxiaohe River and Huangnihe River). DOM in three rivers was composed of protein-like fluorescent (PLF), microbial humus-like fluorescent (MHLF), fulvic-like fluorescent (FLF), and humic-like fluorescent components, which were relative to aromatic groups, phenolic groups, carboxylic groups, and microbial products. The PLF and MHLF were dominated in DOM fractions in the rivers, and the average content of the PLF was the highest in Nanxiaohe River. Humification degree of DOM was the highest in Puhe River, followed by Huangnihe River and Nanxiaohe River. However, molecular mass of DOM in Puhe River was the lowest, followed by Huangnihe River and Nanxiaohe River. Based on the 2D-COS of the SFS and UV-visible spectra, the variation order of DOM fractions in Puhe River was PLF → MHLF → FLF, and the PLF was consistent with the phenolic groups, aromatic groups, and carboxylic groups, but the adverse trend with the microbial products. The variation order in Nanxiaohe River was MHLF → PLF → FLF, and the MHLF was consistent with the aromatic groups, phenolic groups, carboxylic groups, and microbial products. The variation order in Huangnihe River was MHLF → PLF → FLF too, and the PLF was consistent with the carboxylic groups and aromatic groups. The results of the present study demonstrate that UV-visible spectroscopy and SFS combined with 2D-COS are useful methods to characterize structural composition of DOM from urban black and stinky rivers so as to investigate their pollution status.
Due to the emergence of multidrug-resistant pathogens, it is necessary to develop options to fight infections caused by these agents. Lactoferrin (Lf) is a cationic nonheme multifunctional glycoprotein of the innate immune system of mammals that provides numerous benefits. Lf is bacteriostatic and/or bactericidal, can stimulate cell proliferation and differentiation, facilitate iron absorption, improve neural development and cognition, promote bone growth, prevent cancer and exert anti-inflammatory and immunoregulatory effects. Lactoferrin is present in colostrum and milk and is also produced by the secondary granules of polymorphonuclear leukocytes, which store this glycoprotein and release it at sites of infection. Lf is also present in many fluids and exocrine secretions, on the surfaces of the digestive, respiratory and reproductive systems that are commonly exposed to pathogens. Apo-Lf (an iron-free molecule) can be microbiostatic due to its ability to capture ferric iron, blocking the availability of host iron to pathogens. However, apo-Lf is mostly microbicidal via its interaction with the microbial surface, causing membrane damage and altering its permeability function. Lf can inhibit viral entry by binding to cell receptors or viral particles. Lf is also able to counter different important mechanisms evolved by microbial pathogens to infect and invade the host, such as adherence, colonization, invasion, production of biofilms and production of virulence factors such as proteases and toxins. Lf can also cause mitochondrial and caspase-dependent regulated cell death and apoptosis-like in pathogenic yeasts. All of these mechanisms are important targets for treatment with Lf. Holo-Lf (the iron-saturated molecule) can contain up to two ferric ions and can also be microbicidal against some pathogens. On the other hand, lactoferricins (Lfcins) are peptides derived from the N-terminus of Lf that are produced by proteolysis with pepsin under acidic conditions, and they cause similar effects on pathogens to those caused by the parental Lf. Synthetic analog peptides comprising the N-terminus Lf region similarly exhibit potent antimicrobial properties. Importantly, there are no reported pathogens that are resistant to Lf and Lfcins; in addition, Lf and Lfcins have shown a synergistic effect with antimicrobial and antiviral drugs. Due to the Lf properties being microbiostatic, microbicidal, anti-inflammatory and an immune modulator, it represents an excellent natural alternative either alone or as adjuvant in the combat to antibiotic multidrug-resistant bacteria and other pathogens. This review aimed to evaluate the data that appeared in the literature about the effects of Lf and its derived peptides on pathogenic bacteria, protozoa, fungi and viruses and how Lf and Lfcins inhibit the mechanisms developed by these pathogens to cause disease.
Human serum albumin (HSA), one of the most copious plasma proteins is responsible for binding and transportation of many exogenous and endogenous ligands including drugs. In this study, we intended to explore the extent and types of binding interaction present between HSA and the antihypertensive drug, telmisartan (TLM). The conformational changes in HSA due to this binding were also studied using different spectroscopic and molecular docking techniques. The spectral shifting and intensity variations upon interaction with TLM were studied using FT-IR spectroscopy. Binding constant and the change in absorption of HSA at its λmax was analyzed using absorption spectroscopy. Eventually, the types and extent of binding interactions were confirmed using molecular docking technique. Results have shown that TLM significantly interacts with the binding site-1 of HSA utilizing strong hydrogen bonding with Glu292, and Lys195 residues. The UV-absorption intensities were found to be decreased serially as the drug concentration increased with a binding constant of 1.01 x 10³ M⁻¹. The secondary structure analysis using FT-IR spectroscopy also revealed a marked reduction in the α-helix (56%) component of HSA on interaction. This study gives critical insights into the interaction of TLM with HSA protein which eventually affects the concentration of TLM reaching the site of action and ultimately its therapeutic profile.
The human serum albumin (HSA) is produced in the hepatocytes of human livers and is the most abundant protein in blood. HSA consists of one N-terminus, one C-terminus and three homologous domains, each of which has two helical subdomains. HSA enjoys advantages such as long half-life, repeated recycling, and specific accumulation. HSA has several binding pockets where various ligands such as fatty acids, ions can bind to, providing drugs the option for non-covalent binding sites. Meanwhile, two terminal ends of albumin and certain amino acid residues provide more choices for drug targeting. The free amino acid cysteine at the position 34 (Cys34) is located on the surface of albumin and provides a unique thiol group for drugs of interest to be covalently coupled with. The two terminal ends of albumin can be fused with peptides or proteins of interest by molecular recombinant technology. Importantly, HSA can retain its binding affinity in albumin-FcRn interactions after being covalently or non-covalently coupled with various drugs. HSA can also extend the half-life of drugs, reduce renal clearance of drugs and help accumulate drugs in specific tumor tissues. HSA provides a promising opportunity for delivering established medications in a new way.
Antimicrobial peptides in recent years have gained increased interest among scientists, health professionals and the pharmaceutical companies owing to their therapeutic potential. These are low molecular weight proteins with broad range antimicrobial and immuno modulatory activities against infectious bacteria (Gram positive and Gram negative), viruses and fungi. Inability of micro-organisms to develop resistance against most of the antimicrobial peptide has made them as an efficient product which can greatly impact the new era of antimicrobials. In addition to this these peptide also demonstrates increased efficacy, high specificity, decreased drug interaction, low toxicity, biological diversity and direct attacking properties. Pharmaceutical industries are therefore conducting appropriate clinical trials to develop these peptides as potential therapeutic drugs. More than 60 peptide drugs have already reached the market and several hundreds of novel therapeutic peptides are in preclinical and clinical development. Rational designing can be used further to modify the chemical and physical properties of existing peptides. This mini review will discuss the sources, mechanism and recent therapeutic applications of antimicrobial peptides in treatment of infectious diseases.
Donepezil, a highly selective and reversible inhibitor, is amongst the widely used drugs for the treatment of Alzheimer’s disease (AD) across the globe. Clinically significant human transferrin (hTf) is a key player involved in iron metabolism. The current study targets to have an insight into the interaction between donepezil and this plasma protein, hTf using UV–vis absorbance, fluorescence, isothermal titration calorimetry, circular dichroism (CD) and molecular docking techniques. UV spectroscopy of hTf in the presence of donepezil revealed the occurrence of hyperchromism coupled with blue shift in indicating complex formation. Binding constant obtained from UV–vis absorption measurements was of the order 0.9 × 10⁴ M⁻¹ implicative of strong binding. Fluorescence spectroscopy shows that donepezil quenches fluorescence intensity of the native hTf in dynamic manner with a shift in wavelength maxima. Donepezil binds to hTf at 310 K with a binding constant of 1.3 × 10⁴ M⁻¹ suggestive of strong binding. ITC results clearly suggest the reaction to be spontaneous and thermodynamically favorable. CD spectroscopy also showed a shift in the spectrum of the native hTf in the presence of donepezil suggesting formation of complex between protein and drug. Furthermore, molecular docking experiments showed Pro 91, Phe 94 and His 207 to be key players involved in the complex formation. This study gives an insight into the molecular basis of interaction between donepezil and hTf thereby aiding in understanding the activity and mechanism of protein and drug binding. This will serve as a boon for drug designing industries which are working day and night for improved treatment of existing disorders across the world.
Communicated by Ramaswamy H. Sarma
10-Dehydroxyl-12-demethoxy-conophylline is a natural anticancer candidate. The motivation of this study was to explore the pharmacokinetic profiles, tissue distribution, and plasma protein binding of 10-dehydroxyl-12-demethoxy-conophylline in Sprague Dawley rats. A rapid, sensitive, and specific ultra-performance liquid chromatography (UPLC) system with a fluorescence (FLR) detection method was developed for the determination of 10-dehydroxyl-12-demethoxy-conophylline in different rat biological samples. After intravenous (i.v.) dosing of 10-dehydroxyl-12-demethoxy-conophylline at different levels (4, 8, and 12 mg/kg), the half-life t1/2α of intravenous administration was about 7 min and the t1/2β was about 68 min. The AUC0→∞ increased in a dose-proportional manner from 68.478 μg/L·min for 4 mg/kg to 305.616 mg/L·min for 12 mg/kg. After intragastrical (i.g.) dosing of 20 mg/kg, plasma levels of 10-dehydroxyl-12-demethoxy-conophylline peaked at about 90 min. 10-dehydroxyl-12-demethoxy-conophyllinea absolute oral bioavailability was only 15.79%. The pharmacokinetics process of the drug was fit to a two-room model. Following a single i.v. dose (8 mg/kg), 10-dehydroxyl-12-demethoxy-conophylline was detected in all examined tissues with the highest in kidney, liver, and lung. Equilibrium dialysis was used to evaluate plasma protein binding of 10-dehydroxyl-12-demethoxy-conophylline at three concentrations (1.00, 2.50, and 5.00 µg/mL). Results indicated a very high protein binding degree (over 80%), reducing substantially the free fraction of the compound.
Circular dichroism (CD) spectroscopy is a well-established technique for studying the secondary structures, dynamics, folding pathways, and interactions of soluble proteins, and is complementary to the high resolution but generally static structures produced by X-ray crystallography, NMR spectroscopy, and cryo electron microscopy. CD spectroscopy has special relevance for the study of membrane proteins, which are difficult to crystallise and largely ignored in structural genomics projects. However, the requirement for membrane proteins to be embedded in amphipathic environments such as membranes, lipid vesicles, detergent micelles, bicelles, oriented bilayers, or nanodiscs, in order for them to be soluble or dispersed in solution whilst maintaining their structure and function, necessitates the use of different experimental and analytical approaches than those employed for soluble proteins. This review discusses specialised methods for collecting and analysing membrane protein CD data, highlighting where protocols for soluble and membrane proteins diverge.
Interaction between bovine serum albumin (BSA) and phosphorus heterocycles (PHs) was studied using multi-spectroscopic techniques. The results indicated the high binding affinity of PHs to BSA as it quenches the intrinsic fluorescence of BSA. The experimental data suggested the fluorescence quenching mechanism between PHs and BSA as a dynamic quenching. From the UV-vis studies, the apparent association constant (Kapp) was found to be 9.25 ×102, 1.27×104 and 9.01×102 L/mol for the interaction of BSA with pH-1, pH-2 and pH-3 respectively. According to the Förster's non-radiation energy transfer (FRET) theory, the binding distances between BSA and PHs have been calculated. The binding distances (r) of pH-1, pH-2 and pH-3 were found to be 2.86, 3.03, 5.12 nm, respectively indicating energy transfer occurs between BSA and PHs. The binding constants of the PHs obtained from the fluorescence quenching data were found to be decreased with increase of temperature. The negative values of the thermodynamic parameters ΔH, ΔS and ΔG at different temperatures revealed that the binding process is spontaneous; hydrogen bonds and van der Waals interaction were the main force to stabilize the complex. The micro-environment of the protein-binding site was studied by synchronous fluorescence and circular dichroism (CD) techniques and data indicated that the conformation of BSA changed in the presence of PHs. Finally, we studied the BSA-PHs docking using auto dock and results suggest that PHs is located in the cleft between the domains of BSA.
The more than 100.000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic “artifacts”, including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density map, thus limiting the relevance of structure determinations. Moreover, for most of these structures no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in the inference for protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5’-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms.
Steady state and time resolved fluorescence spectroscopy, combined with molecular dynamics simulation, have been used to explore the interactions of a therapeutically important bioflavonoid, genistein, with normal human hemoglobin (HbA). Binding constants estimated from the fluorescence studies were K = (3.5 0.32) 10 4 M −1 for genistein. Specific interactions with HbA were confirmed from flavonoid-induced fluorescence quenching of the tryptophan in the protein HbA. The mechanism of this quenching involves both static and dynamic components as indicated by: (a) increase in the values of Stern-Volmer quenching constants with temperatures, (b) 0 / is slightly > 1 (where 0 and are the unquenched and quenched tryptophan fluorescence lifetimes (averaged) respectively). Molecular docking and dynamic simulations reveal that genistein binds between the subunits of HbA, ~18 Å away from the closest heme group of chain α1, emphasizing the fact that the drug does not interfere with oxygen binding site of HbA.
The interaction between bovin β-Lactoglobulin (β-LG) and retinol at two different pH values was investigated by multispectroscopic, zeta potential, molecular modeling, and conductometry measurements. The steady state and polarization fluorescence spectroscopy revealed that complex formation at two different pH values could occur through a remarkable static quenching. According to fluorescence quenching, one set of binding site at pH 2 and two sets of binding sites at pH 7 were introduced for binding of retinol to β-LG that show the enhancement of saturation score of β-LG to retinol in dimmer condition. The polarization fluorescence analysis represented that there is more affinity between β-LG and retinol at pH 7 rather than at pH 2. The effect of retinol on β-LG was studied by UV-visible, circular dichroism (CD), and synchronous fluorescence, which indicated that retinol induced more structural changes on β-LG at pH 7. β-LG-retinol complex formation at two different pH values was recorded via applying resonance light scattering (RLS) and zeta potential. Conductometry and RLS showed two different behaviors of interaction between β-LG and retinol at two different pH values; therefore, dimmer formation played important roles in different behaviors of interaction between β-LG and retinol. The zeta potential was the implied combination of electrostatic and hydrophobic forces which are involved in β-LG-retinol complex at two different pH values, and the hydrophobic interactions play a dominant role in complex formation. Molecular modeling was approved by all experimental results. The acquired results suggested that monomer and dimmer states of β-LG can be induced by retinol with different behaviors.
This paper describes the interaction between 2,4-dinitrophenol (DNP) with the two drug carrier proteins - human serum albumin (HSA) and human holo transferrin (HTF). Hence, binding characteristics of DNP to HSA and HTF were analyzed by spectroscopic and molecular modeling techniques. Based on results obtained from fluorescence spectroscopy, DNP had a strong ability to quench the intrinsic fluorescence of HSA and HTF through a static quenching procedure. The binding constant and the number of binding sites were calculated as 2.3 × 10(11) M(-1) and .98 for HSA, and 1.7 × 10(11) M(-1) and 1.06 for HTF, respectively. In addition, synchronous fluorescence results showed that the microenvironment of Trp had a slight tendency of increasing its hydrophobicity, whereas the microenvironment of the Tyr residues of HSA did not change and that of HTF showed a significant trend (red shift of about 4 nm) of an increase in polarity. The distance between donor and acceptor was obtained by the Förster energy according to fluorescence resonance energy transfer, and was found to be 3.99 and 3.72 nm for HSA and HTF, respectively. The critical induced aggregation concentration (CCIAC) of the drug on both proteins was determined and confirmed by an inflection point of the zeta potential behavior. Circular dichroism data revealed that the presence of DNP caused a decrease of the α-helical content of HSA and HTF, and induced a remarkable mild denaturation of both proteins. The molecular modeling data confirmed our experimental results. This study is deemed useful for determining drug dosage.
Chemical, physical, biological and materials engineering disciplines use a variety of chiroptical spectroscopies to probe geometrical and optical asymmetry in molecules and particles. Electronic (ECD) and vibrational (VCD) circular dichroism are the most common of these techniques and collectively enable the studies of electronic and vibronic transitions with energies between 0.1 and 5.0 eV. The vibrational states with characteristic energies in the range of 0.001-0.01 eV carry valuable information about concerted intermolecular motions in molecules and crystals involving multiple atoms. These vibronic transitions located in the terahertz (THz) part of the spectrum become increasingly more important for the chemistry, physics, and biology of complex molecules and materials However, the methodology and hardware of THz circular dichroism (TCD) are much less developed than the chiroptical spectroscopies for ultraviolet, visible, near- and mid infrared photons. Here we provide theoretical foundations, practical implementations, comparative assessments, and exemplary applications of TCD spectroscopy. We show that the sign, intensity, and position of TCD peaks are highly sensitive to the three-dimensional structure and long-range organization of molecular crystals, which offer unique capabilities to study (bio) molecules, their crystals, and nanoscale assemblies and apply the novel data processing methodologies. TCD also offers a convenient toolbox to identify new physical phenomena, such as chiral phonons and their propagation in nanostructured matter. We also discuss the major challenges, emerging opportunities and promising research directions, including broad investigation of chiral phonons observed in chiral (nano) crystals and emerging machine learning methodologies for TCD in biological and nanoscale structures. Ubiquity of low-frequency vibrations with rotational components in biomolecular structures, combined with sharpness of peaks in TCD spectra, enables a variety of technological translations.
Excessive iron deposition is linked to the pathology of neurodegenerative, signifying the importance of iron homeostasis. Together with ferritin, human transferrin (hTf) is at the heart of iron homeostasis. Naringenin (Nag) is a flavanone known to have neuroprotective action. Inhibition of hTf, which reduces the free iron, is an attractive strategy to treat neurodegenerative diseases. This study explores the binding of nag to hTf employing in-silico and in vitro approaches. Molecular docking studies revealed that nag binds to hTf with a high affinity through metal coordination bonding. Further, 200 ns all-atom molecular dynamic simulation studies were performed to understand the hTf-Nag complex dynamics. It was found that no significant structural alterations occur in hTf upon binding of nag, suggesting the stability of the hTf-Nag complex. Computational studies were supported by experimental assays. Fluorescence binding studies suggested that Nag binds to hTf with an excellent affinity. Isothermal titration calorimetric (ITC) studies further validated the spontaneous nature of binding of nag to hTf and provided various thermodynamic parameters of the hTf-Nag complex. This study provides a platform to use nag in therapeutic strategies to manage neurodegenerative disorders.
Background
Von Willebrand Factor (VWF) is a multimeric protein largely involved in both primary and secondary hemostasis. The diagnosis and classification of von Willebrand Disease (VWD) patients can be challenging. In this poster we explore the genetic defects and their structural consequences in a VWD patient with a disproportionately high bleeding phenotype for her VWD severity. A 31-year old female, initially diagnosed with VWD type 1, presented herself with a bleeding tendency (ISTH-BAT 13) disproportionate to the severity of her VWD with antigen levels of 36%. Additional analysis showed decreased FVIII-binding at 28%. The combination of both quantitative and functional defects of VWF was an indication for further genetic analysis in order to better define the subtype of VWD.
Aims
Genetic and structural analysis of VWF in a patient with a disproportionally high bleeding phenotype with regard to a mild decrease in VWF antigen.
Methods
Routine laboratory analysis for VWD was performed. Genetic screening was performed by exome sequencing of hemostasis related genes. VWF mRNA analysis was carried out by RT-PCR and Sanger sequencing. The X-ray structure of furin in complex with a peptide-based inhibitor (PDB ID: 6YD7) was used as a template to construct furin-VWF (759HR(R760S)SKRS764) complex. The derived structures (furin in complex with WT/R760S-VWF) were subjected to molecular dynamics (MD) simulations (200ns) and binding free energy (BFE) calculations by using standard parameters and protocols implemented in AMBER20 program.
Results
Routine analysis showed PFA-ADP and PFA EPI >300 seconds, VWF:ACT of 37% with a VWF:AG of 36%. Collagen binding and FVIII-binding were 46% and 28% respectively. Genetic analysis of the VWF gene disclosed 2 heterozygous variants of unknown significance (VUS): c.2771 G>A (exon 21, p.Arg924Gln) has a 1-2.5% population prevalence and has been previously described in type 1 and 2N VWD. The other VUS (c.2278 C>A; exon 17) is a novel mutation predicting a major amino acid substitution (p.Arg760Ser) in the D2-domain of VWF. Sequencing of exons 17 and 21 in the patient's VWF mRNA revealed homozygosity for the mutated allele at both mutation sites, indicating that the two variants are in cis and that the 'normal' allele is not expressed at mRNA level. Molecular dynamics simulations of the novel c.2278 C>A mutation (Arg760Ser) predicts a markedly decreased binding of furin to its VWF binding site, possibly decreasing or preventing VWF pro-peptide cleavage. This in turn has been shown to lead to reduced FVIII-binding of VWF.
Conclusion
Genetic analysis shows one polymorphism (c.2771 G>A) and one variation of unknown significance (c.2278 C>A) in the patient's VWF-gene. The polymorphism is known to be of low pathogenicity. The c. 2278 C>A mutation was not known in any of the mutation databases and is a novel VWF mutation. Both mutations were shown
to be present on the same allele. As the wild-type allele was not expressed on mRNA level, all of the patient's VWF protein includes both amino acid substitutions. Modeling and molecular dynamics simulations show a markedly decreased affinity of furin to its cleavage site on the VWF protein due to the Arg760Ser substitution, likely resulting in a persistent pro-peptide binding to the mature VWF protein.
Figure 1 Figure 1.
Disclosures
No relevant conflicts of interest to declare.
Hemoglobin, a homodimeric globular protein, is found predominantly in red blood cells and in a small amount in blood plasma. Along with binding to certain native molecules, it also interacts with various xenobiotics. The present review aims at studying these interactions and the resultant tangible impact on the structure and function of the protein if any. The review also encompasses various analytical and computational approaches which are routinely used to study these interactions. A detailed discussion on types of interaction exhibited by individual xenobiotics has been included herein. Additionally, the effects of xenobiotic binding on the oxygen carrying capacity of hemoglobin have been reviewed. These insights would be of great value in drug design and discovery. Envisaging probable interactions of designed ligands with hemoglobin would help improvise the process of drug development. This would also open up new avenues for studying hemoglobin-mediated drug delivery.
In this work, we investigated the simultaneous binding of curcumin (CUR) to human serum albumin (HSA) and human-holo transferrin (HTF) in the roles of binary and ternary systems. The binding affinity and binding site of protein-protein interaction were studied by the methods of multiple spectroscopic and molecular dynamics (MD) simulation. According to the results, the measurements for binding constant of HSA-CUR, HTF-CUR and (HSA-HTF) CUR complexes were observed to be 1.51 × 10⁵, 7.93 × 10⁴ and 1.44 × 10⁵ M⁻¹ respectively. Thermodynamic parameters were considered to be set at three varying temperatures including 298, 303, and 308 K. In conformity to the negative values of ΔH⁰ and ΔS⁰ the significant roles of hydrogen binding and van der-Waals forces in the formation of complexes are quiet evident. The binding distance between Trp residues of HSA, HTF and HSA-HTF upon interaction with CUR, were acquired by applying the Förster’s theory of non-radioactive energy transfer and reported to be 2.04 nm, 1.78 nm, and 1.86 nm, respectively. In accordance with the conductometry and Resonance light scattering (RLS) results, there were different interaction behaviors among the HSA-HTF complex and CUR in ternary system when being compared to the outcomes of binary system. The secondary structure of all three cases increased as the CUR concentration was intensified, which confirmed the inducement of proteins conformational changes through the application of circular dichroism (CD) technique. The experimental results that were acquired throughout the binding of HSA-CUR, HTF-CUR, and (HSA-HTF) CUR complexes were approved by molecular modeling.
Communicated by Ramaswamy H. Sarma
During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cell biological systems under physiological conditions. Here, we review key technological improvements that have established AFM as an analytical tool to observe and quantify native biological systems from the micro- to the nanoscale. Native biological systems include living tissues, cells, and cellular components such as single or complexed proteins, nucleic acids, lipids, or sugars. We showcase the procedures to customize nanoscopic chemical laboratories by functionalizing AFM tips and outline the advantages and limitations in applying different AFM modes to chemically image, sense, and manipulate biosystems at (sub)nanometer spatial and millisecond temporal resolution. We further discuss theoretical approaches to extract the kinetic and thermodynamic parameters of specific biomolecular interactions detected by AFM for single bonds and extend the discussion to multiple bonds. Finally, we highlight the potential of combining AFM with optical microscopy and spectroscopy to address the full complexity of biological systems and to tackle fundamental challenges in life sciences.
Engineering protein and peptide-based materials for drug delivery applications has gained momentum due to their biochemical and biophysical properties over synthetic materials, including biocompatibility, ease of synthesis and purification, tunability, scalability, and lack of toxicity. These biomolecules have been used to develop a host of drug delivery platforms, such as peptide- and protein-drug conjugates, injectable particles, and drug depots to deliver small molecule drugs, therapeutic proteins, and nucleic acids. In this review, we discuss progress in engineering the architecture and biological functions of peptide-based biomaterials -naturally derived, chemically synthesized and recombinant- with a focus on the molecular features that modulate their structure-function relationships for drug delivery.
Concern over antibiotic resistance is growing, and new classes of antibiotics, particularly against Gram-negative bacteria, are needed. Antimicrobial peptides (AMPs) have been proposed as a new class of clinically useful antimicrobials. Special attention has been devoted to frog-skin temporins. In particular, temporin L (TL) is strongly active against Gram-positive, Gram-negative bacteria and yeast strains. With the aim of overcoming some of the main drawbacks preventing the widespread clinical use of this peptide, i.e. toxicity and unfavorable pharmacokinetics profile, we designed new formulations combining TL with different types of cyclodextrins (CDs). TL was associated to a panel of neutral or negatively charged, monomeric and polymeric CDs. The impact of CDs association on TL solubility, as well as the transport through bacterial alginates was assessed. The biocompatibility on human cells together with the antimicrobial and antibiofilm properties of TL/CD systems was explored.
Background and objective:
Human serum albumin (HSA) controls the flow of numerous chemical structures and molecules in the cardiovascular system. So, thermal conductivity of this atomic compound is important in medicinal applications.
Methods:
In this work, the thermal conductivity of HSA is calculated with equilibrium/non-equilibrium molecular dynamic approaches. In these methods each HSA molecule is exactly represented by C, N, O and S atoms and their implemented dreiding potential. Finally by using Green-Kubo and Fourier's law the thermal conductivity of HSA/H2O mixture is calculated.
Results:
Our calculated rates for thermal conductivity via equilibrium/non-equilibrium molecular dynamics methods are 0.496 W/m K and 0.448 W/m K, respectively. The calculated thermal conductivity for this structure was very close to the thermal conductivity calculated for water molecules which were reported by other research groups. Furthermore our simulated structures show that thermal conductivity of HAS/H2O mixtures has inverse relation with HAS molecules numbers and temperature of simulated atomic structures.
Conclusions:
Comparing thermal conductivity from equilibrium/non-equilibrium molecular dynamics methods for HAS/H2O shows that EMD and NEMD results are reliable and EMD calculated results are higher than NEMD results.
Sample inner filter effect (IFE) induces spectral distortion and affects the linearity between intensity and analyte concentration in fluorescence, Raman, surface enhanced Raman, and Rayleigh light scattering measurements. Existing spectrofluorometeric-based measurements treat the light scattering and absorption identically in their sample IFEs. Reported herein is the finding that photon scattering and absorption differ drastically in inducing the sample IFE in Stokes-shifted fluorescence (SSF) spectrum, resonance synchronous spectrum (RS2), and the polarized resonance synchronous spectrum (PRS2) measurements. Absorption with an absorption extinction as small as 0.05 imposes significant IFE on SSF, RS2, and PRS2 measurements. However, no significant IFE occurs even when the scattering extinction is as high as 0.9. For samples that both absorb and scatter light, one should decompose their UV-vis extinction spectra into absorption and scattering extinction component spectra before correcting the sample IFE. An iteration PRS2 method was introduced for the experimental decoupling the photon absorption and scattering contribution.The methodology presented in this work can be easily implemented by researchers with access to one conventional UV-vis spectrophotometer and one spectrofluorometers equipped with pair of excitation and detection polarizers. This work should be of broad significance in chemical research given the popularity of fluorescence spectroscopy in material characterization applications.
Macroscopic fluorescence lifetime imaging (MFLI) has been proved to be an accurate tool to quantify FRET lifetime‐based assessment of receptor‐ligand engagement in vitro and in vivo. Herein, we report on the quantitative comparison of MFLI for whole‐body preclinical studies in transmittance and reflectance geometries. The comparative study was conducted both for in vitro and in vivo conditions. FRET quantification performance in both geometries was similar in high fluorescence concentration samples. However, the reflectance geometry performed better at low fluorescence concentration. In addition, reflectance geometry could acquire sub‐surface imaging of the main whole‐body organs of small animals without being compromised by tissue attenuation.
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Temporin A (FLPLIGRVLSGIL-NH2), temporin F (FLPLIGKVLSGIL-NH2), and temporin G (FFPVIGRILNGIL-NH2), first identified in skin secretions of the frog Rana temporaria, produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal β-cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca2+ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration-dependent stimulation of insulin release from 1.1B4 human-derived pancreatic β-cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon-like peptide-1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.
We explore the mechanism of fluorescence enhancement and fluorescence quenching effect of single walled carbon nanotubes (SWCNTs) on highly fluorescent graphene quantum dot (GQDs) over a wide range of concentrations of SWCNTs. At very low concentrations of SWCNTs, fluorescence intensity of GQDs is enhanced, while at higher concentrations, systematic quenching of fluorescence is observed. The nature of Stern–Volmer plot for the later case was found to be non-linear indicating combined effect of dynamic and static quenching. The contribution of dynamic quenching component was assessed through the fluorescence lifetime measurements. The contribution of static quenching is confirmed from the red shift of fluorescence spectra of the GQDs after addition of SWCNTs. The fluorescence intensity is first enhanced at very low concentration due to improved dispersion and higher absorption by GQDs, while at higher concentration the fluorescence of GQDs is quenched due to the complex formation and associated reduction of the radiative sites of GQDs, which is confirmed from time resolved fluorescence measurements. Laser confocal microscopy imaging provides direct evidence of the enhancement and quenching of fluorescence at low and high concentration of SWCNTs, respectively. This study provides an important insight in tuning the fluorescence of GQDs and understanding the interaction between GQDs and different CNTs, which is important for bio-imaging and drug delivery applications.
Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O2 or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the β subunits. However, these changes are not observed in the isolated β chain (β4 homotetramer) and, therefore, are associated with the α–β inter-subunit interactions. For the communications between α and β subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O2 binding mechanism of Hb A.
The interaction between apo-human serum transferrin (Apo-hTf) and alprazolam was investigated using various spectroscopic techniques. The drug quenched the fluorescence intensity of Apo-hTf and the mechanism behind the quenching was static. The thermodynamic parameters (ΔG, ΔH, and ΔS) that obtained from tryptophan fluorescence study revealed that the interactions between alprazolam and Apo-hTf were spontaneous. Collectively, hydrophobic interactions and hydrogen bonding most likely played major roles in Apo-hTf/alprazolam interactions. Also, the absorption spectra of Apo-hTf increased in the presence of increasing concentration of alprazolam, reflecting Apo-hTf structural alteration after drug's binding. The CD results demonstrated that the Apo-hTf/alprazolam interaction does not affect the protein secondary and tertiary structure significantly until the molar ratios (alprazolam/Apo-hTf) of 10, but the conformational changes become visible at higher molar ratios. The DSC results suggested that alprazolam stabilized the Apo-hTf at alprazolam/Apo-hTf molar ratio of 20. Based on the achieved results, this potentially therapeutic agent can significantly bind to Apo-hTf which also further confirmed by molecular docking study. This study on the interaction of the drug with Apo-hTf should be helpful for understanding the transportation and distribution of drugs in vivo, as well as the action mechanism and dynamics of a drug at the molecular level.
The interaction between juglone at the concentration range of 10–110 µM and bovine serum albumin (BSA) or human serum albumin (HSA) at the constant concentration of 11 µM was investigated by fluorescence and UV absorption spectroscopy under physiological-like condition. Performing the experiments at different temperatures showed that the fluorescence intensity of BSA/HSA was decreased in the presence of juglone by a static quenching mechanism due to the formation of the juglone–protein complex. The binding constant for the interaction was in the order of 10³ M⁻¹, and the number of binding sites for juglone on serum albumins was determined to be equal to one. The thermodynamic parameters including enthalpy (ΔH), entropy (ΔS) and Gibb’s free energy (ΔG) changes were obtained by using the van’t Hoff equation. These results indicated that van der Waals force and hydrogen bonding were the main intermolecular forces stabilizing the complex in a spontaneous association reaction. Moreover, the interaction of BSA/HSA with juglone was verified by UV absorption spectra and molecular docking. The results of synchronous fluorescence, UV–visible and CD spectra demonstrated that the binding of juglone with BSA/HSA induces minimum conformational changes in the structure of albumins. The increased binding affinity of juglone to albumin observed in the presence of site markers (digoxin and ibuprofen) excludes IIA and IIIA sites as the binding site of juglone. This is partially in agreement with the results of molecular docking studies which suggests sub-domain IA of albumin as the binding site.
The root mean square deviation (RMSD) is a similarity measure widely used in analysis of macromolecular structures and dynamics. As increasingly larger macromolecular systems are being studied, dimensionality effects such as the “curse of dimensionality” (a diminishing ability to discriminate pairwise differences between conformations with increasing system size) may exist and significantly impact RMSD-based analyses. For such large bimolecular systems, whether the RMSD or other alternative similarity measures might suffer from this “curse” and lose the ability to discriminate different macromolecular structures had not been explicitly addressed. Here, we show such dimensionality effects for both weighted and non-weighted RMSD schemes. We also provide a mechanism for the emergence of the “curse of dimensionality” for RMSD from the law of large numbers by showing that the conformational distributions from which RMSDs are calculated become increasingly similar as the system size increases. Our findings suggest the use of weighted RMSD schemes for small proteins (less than 200 residues) and non-weighted RMSD for larger proteins when analyzing molecular dynamics trajectories.
Aim:
MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages.
Methods:
Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3'-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip).
Results:
LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability.
Conclusion:
miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases.
Transferrin (Tf) is an essential transport protein circulating iron in the blood and delivering to tissues. It displays highly pH dependent cooperativity between the two lobes each carrying an iron, and forms a tight complex with the receptor during endocytosis and recycling back to the serum. We explore short-term dynamics within selected microstates of human Tf to identify functional information relevant to long-term dynamics. While the variance-covariance matrix delineates cooperativity between the domains of Tf at serum pH which is lost at endosomal pH, its decomposition does not bring about additional information. We employ perturbation-response scanning (PRS) to extract essential components that contribute to a pre-selected conformational change. Since large-scale motions may require key residues to mediate correlated motions between different regions of the protein, we use PRS to predict those involved in the conformational transitions between the iron bound and free hTf. Physiological and endosomal conditions are mimicked to identify critical residues for holo→apo and apo→holo transitions. Iron binding motions are mainly orchestrated by residues at the synergistic anion uptake sites, a finding also corroborated by additional molecular dynamics simulations where these sites are perturbed by docking the anion. Iron release is not readily accessible at serum pH, while at endosomal pH single residue perturbations on any residue encourage the large transition that involves a complex twisting of the two domains relative to each other, simultaneously opening both lobes. pH dependent change in the dynamics is traced to the altered electrostatic potential distribution along the surface. Examination of local dynamics in hTf-receptor pair reveals cooperativity in the quaternary structure and explains resistance to iron release in the complex. Meanwhile, analysis of hTf complex with a bacterial receptor that has evolved to sequester iron identifies two regions contacting rapidly evolving residues that mechanically manipulate dissociation from the pathogen.
Antimicrobial Peptides (AMPs) are an organism's built-in defence molecules that have attracted extensive research attention worldwide. Harnessing and creating them synthetically has the potential to help overcome increasing antibiotic resistance in many pathogens. In addition to covering the current advances in AMP research, this volume examines new technologies such as bioinformatics, combinatorial libraries, high-throughput screening, peptidomimetics, biophysics, and structural biology. This volume also describes new methods and strategies for AMP prediction, design, and applications that overcome obstacles in developing them into therapeutic agents.
A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.
The motions of a dye-labeled DNA hairpin loop (Cy5-5'-GGGTT-(A)(30)-AACCC-3'-TMR) have been investigated through the fluctuations in proximity ratio from fluorescence resonance energy transfer (FRET). We examine three solution conditions: (1) MilliQ water, (2) Tris-EDTA buffer, and (3) Tris-EDTA buffer plus an excess of DNA complementary to the loop sequence, (T)(30). Correlations in proximity ratio show submillisecond dynamics. Static heterogeneity is revealed from the distribution of proximity ratio amplitudes. The observed stretched exponential kinetics are consistent with a model based on the transition between two states over a complex energy landscape.
Several spectroscopic approaches namely fluorescence, time-resolved fluorescence, UV-visible, and Fourier transform infra-red (FT-IR) spectroscopy were employed to examine the interaction between ethane-1,2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy)dichloride (16-E2-16) and bovine serum albumin (BSA). Fluorescence studies revealed that 16-E2-16 quenched the BSA fluorescence through a static quenching mechanism, which was further confirmed by UV-visible and time-resolved fluorescence spectroscopy. In addition, the binding constant and the number of binding sites were also calculated. The thermodynamic parameters at different temperatures (298 K, 303 K, 308 K and 313 K) indicated that 16-E2-16 binding to BSA is entropy driven and that the major driving forces are electrostatic interactions. Decrease of the α-helix from 53.90 to 46.20% with an increase in random structure from 22.56 to 30.61% were also observed by FT-IR. Furthermore, the molecular docking results revealed that 16-E2-16 binds predominantly by electrostatic and hydrophobic forces to some residues in the BSA sub-domains IIA and IIIA. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
The binding of nobiletin to human serum albumin (HSA) was investigated by fluorescence, UV–vis, FT-IR, CD, and molecular modeling. Fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K) at four different temperatures (289, 296, 303 and 310 K) were 4.054, 4.769, 5.646 and 7.044×104 M−1, respectively. The enthalpy change (ΔH0) and the entropy changes (ΔS0) were calculated to be 1.938 kJ mol−1 and 155.195 J mol−1 K−1 according to the Van’t Hoff equation. The binding average distance, r, between the donor (HSA) and the acceptor (nobiletin) was evaluated and found to be 2.33 nm according to the Förster's theory of non-radiation energy transfer. Changes in the CD and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. Computational mapping of the possible binding sites of nobiletin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.
Aims:
Temporin-1CEa, a 17-residue antimicrobial peptide, is known to exert broad-spectrum anticancer activity that acts preferentially on cancer cells instead of normal cells. However, the mechanism of cancer cell death induced by temporin-1CEa is weakly understood.
Main methods:
Here, we investigated the cytotoxic and membrane-disrupting effects of temporin-1CEa on human breast cancer cell line Bcap-37, using MTT assay, electronic microscope observation, fluorescence imaging and flow cytometry analysis.
Key findings:
The MTT assay indicated that one-hour temporin-1CEa treatment led to rapid cell death in either caspase-dependent or -independent manner. The electronic microscope observation suggested that temporin-1CEa exposure resulted in profound morphological changes in Bcap-37 cells. The fluorescence imaging and flow cytometry analysis demonstrated that temporin-1CEa exhibited membrane-disrupting property characterized by induction of cell-surface phosphatidylserine exposure, elevation of plasma membrane permeability, and rapid transmembrane potential depolarization. Moreover, temporin-1CEa might also induce rapid cell death through mitochondria-involved mechanisms, including rapid intracellular Ca(2+) leakage, collapse of mitochondrial membrane potential (Δφm) and over-generation of reactive oxygen species (ROS).
Significance:
In summary, the present study indicates that temporin-1CEa triggers a rapid cytotoxicity in Bcap-37 cells through membrane-destruction and intracellular mechanisms involving mitochondria. These intracellular mechanisms and direct membrane-destruction effect were evaluated helping to understand the detail action of antimicrobial peptides in mammalian cancer cells.