Interfacial Glucose to Regulate Hydrated Lipid Bilayer Properties: Influence of Concentrations.

A series of atomistic molecular dynamics (MD) simulations were carried out with a hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer with the variation of glucose concentrations from 0 to 30 wt % in the presence of 0.3 M NaCl. The study suggested that although the thickness of the lipid bilayer dropped significantly with the increase in glucose concentration, it expanded laterally at high glucose levels due to the intercalation of glucose between the headgroups of adjacent lipids. We adopted the surface assessment via the grid evaluation method to compute the deviation of the bilayer's key structural features for the different amounts of glucose present. This suggested that the accumulation of glucose molecules near the headgroups influences the local lipid bilayer undulation and crimping of the lipid tails. We find that the area compressibility modulus increases with the glucose level, causing enhanced bilayer rigidity arising from the slow lateral diffusion of lipids. The restricted lipid motion at high glucose concentrations controls the sustainability of the curved bilayer surface. Calculations revealed that certain orientations of CO→ of interfacial glucose with the PN→ of lipid headgroups are preferred, which helps the glucose to form direct hydrogen bonds (HBs) with the lipid headgroups. Such lipid-glucose (LG) HBs relax slowly at low glucose concentrations and exhibit a higher lifetime, whereas fast structural relaxation of LG HBs with a shorter lifetime was noticed at a higher glucose level. In contrast, lipid-water (LW) HBs exhibited a higher lifetime at a higher glucose level, which gradually decreased with the glucose level lowering. The study interprets that the glucose concentration-driven LW and LG interactions are mutually inclusive. Our detailed analysis will exemplify small saccharide concentration-driven membrane stabilizing efficiency, which is, in general, helpful for drug delivery study.

Influence of Aqueous Arginine Solution on Regulating Conformational Stability and Hydration Properties of the Secondary Structural Segments of a Protein at Elevated Temperatures: A Molecular Dynamics Study.

The effects of aqueous arginine solution on the conformational stability of the secondary structural segments of a globular protein, ubiquitin, and the structure and dynamics of the surrounding water and arginine were examined by performing atomistic molecular dynamics (MD) simulations. Attempts have been made to identify the osmolytic efficacy of arginine solution, and its influence in guiding the hydration properties of the protein at an elevated temperature of 450 K. The similar properties of the protein in pure water at elevated temperatures were computed and compared. Replica exchange MD simulation was performed to explore the arginine solution's sensitivity in stabilizing the protein conformations for a wide range of temperatures (300-450 K). It was observed that although all the helices and strands of the protein undergo unfolding at elevated temperature in pure water, they exhibited native-like conformational dynamics in the presence of arginine at both ambient and elevated temperatures. We find that the higher free energy barrier between the folded native and unfolded states of the protein primarily arises from the structural transformation of α-helix, relative to the strands. Our study revealed that the water structure around the secondary segments depends on the nature of amino acid compositions of the helices and strands. The reorientation of water dipoles around the helices and strands was found hindered due to the presence of arginine in the solution; such hindrance reduces the possibility of exchange of hydrogen bonds that formed between the secondary segments of protein and water (PW), and as a result, PW hydrogen bonds take longer time to relax than in pure water. On the other hand, the origin of slow relaxation of protein-arginine (PA) hydrogen bonds was identified to be due to the presence of different types of protein-bound arginine molecules, where arginine interacts with the secondary structural segments of the protein through multiple/bifurcated hydrogen bonds. These protein-bound arginine formed different kinds of bridged PA hydrogen bonds between amino acid residues of the same secondary segments or among multiple bonds and helped protein to conserve its native folded form firmly.

Predicting the evolution of number of native contacts of a small protein by using deep learning approach.

Native contacts (NCs) are one of the most vital parameters in order to define the resemblance of a protein conformation with its native state. Prediction of number of native contacts in a protein is useful in protein folding mechanism. In this work, we focused to predict the time series of the number of NCs of a small protein, insulin monomer by using three neural network based models, namely; Multi-Layer Perceptron (MLP), Long Short Term Memory (LSTM) and Gated Recurrent Unit (GRU). The input data used in the study was the time evolution of NC values of the folded and unfolded protein conformations computed from the equilibrated trajectories of atomistic molecular dynamics (MD) simulations performed with the aqueous solution of the protein at ambient as well as at an elevated temperature. The evolutionary prediction accuracy of the three models was tested by calculating two error parameters; Root Mean Square Error (RMSE) and Mean Absolute Error (MAE). Our study revealed that, although these three models are successful in forecasting the time evolutions of the NCs in terms of lower RMSE and MAE, the prediction through static memoryless artificial neural network, MLP was relatively less precise as compared to other two recurrent units, LSTM and GRU. The study infers that by using the available input data generated from the MD trajectories; these neural network based models could be used to predict the complex evolution pattern of distanced based structural parameters of a protein with a satisfactory level.

Conformational changes of GDNF-derived peptide induced by heparin, heparan sulfate, and sulfated hyaluronic acid - Analysis by circular dichroism spectroscopy and molecular dynamics simulation.

Glial-cell-line-derived neurotrophic factor (GDNF) is a protein that has therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. The activity of GDNF is highly dependent on the interaction with sulfated glycans which bind at the N-terminus consisting of 19 residues. Herein, we studied the influence of different glycosaminoglycan (i.e., glycan; GAG) molecules on the conformation of a GDNF-derived peptide (GAG binding motif, sixteen amino acid residues at the N-terminus) using both experimental and theoretical studies. The GAG molecules employed in this study are heparin, heparan sulfate, hyaluronic acid, and sulfated hyaluronic acid. Circular dichroism spectroscopy was employed to detect conformational changes induced by the GAG molecules; molecular dynamics simulation studies were performed to support the experimental results. Our results revealed that the sulfated GAG molecules bind strongly with GDNF peptide and induce alpha-helical structure in the peptide to some extent.

Analyzing the driving forces of insulin stability in the basic amino acid solutions: A perspective from hydration dynamics.

Amino acids having basic side chains, as additives, are known to increase the stability of native-folded state of proteins, but their relative efficiency and the molecular mechanism are still controversial and obscure as well. In the present work, extensive atomistic molecular dynamics simulations were performed to investigate the hydration properties of aqueous solutions of concentrated arginine, histidine, and lysine and their comparative efficiency on regulating the conformational stability of the insulin monomer. We identified that in the aqueous solutions of the free amino acids, the nonuniform relaxation of amino acid-water hydrogen bonds was due to the entrapment of water molecules within the amino acid clusters formed in solutions. Insulin, when tested with these solutions, was found to show rigid conformations, relative to that in pure water. We observed that while the salt bridges formed by the lysine as an additive contributed more toward the direct interactions with insulin, the cation-π was more prominent for the insulin-arginine interactions. Importantly, it was observed that the preferentially more excluded arginine, compared to histidine and lysine from the insulin surface, enriches the hydration layer of the protein. Our study reveals that the loss of configurational entropy of insulin in arginine solution, as compared to that in pure water, is more as compared to the entropy loss in the other two amino acid solutions, which, moreover, was found to be due to the presence of motionally bound less entropic hydration water of insulin in arginine solution than in histidine or lysine solution.

Unraveling the origin of interactions of hydroxychloroquine with the receptor-binding domain of SARS-CoV-2 in aqueous medium.

Interactions of hydroxychloroquin (HCQ) with the receptor binding domain (RBD) of SARS-CoV-2 were studied from atomistic simulation and ONIOM techniques. The key-residues of RBD responsible for the human transmission are recognized to be blocked in a heterogeneous manner with the favorable formation of key-residue:HCQ (1:1) complex. Such heterogeneity in binding was identified to be governed by the differential life-time of the hydrogen bonded water network anchoring HCQ and the key-residues. The intermolecular proton transfer facilitates the most favorable Lys417:HCQ complexation. The study demonstrates that off-target bindings of HCQ need to be minimized to efficiently prevent the transmission of SARS-CoV-2.

Insights into the Sensitivity of Arginine Concentration to Preserve the Folded Form of Insulin Monomer under Thermal Stress.

Arginine, although popularly known as aggregation suppressor additive, has been found to quench proteins' structure and function by destabilizing their conformations. Driven by such controversial evidence, in this work we performed a series of atomistic molecular dynamics simulations of insulin monomer, a biologically active hormone protein, in arginine solution of varying concentrations (0.5, 1, and 2 M) at ambient and elevated temperature (400 K) to explore the arginine concentration driven structure-based stability of the protein. Our study reveals that the flexibility of the protein's structure is dependent on the arginine concentration, and among all the used solutions, 2 M arginine, a "neutral crowder" that mimics the cellular environment, can preserve the native folded form of the protein at ambient temperature in an excellent manner. Further, while the protein unfolds at 400 K in pure water, this solution worked satisfactorily to preserve the protein's folded conformation more firmly than the other solutions. The replica-exchange MD of insulin in 2 M arginine solution further supports the fact. In this aspect an important issue in molecular pharmacology is to identify and recognize the physical origin of the stability of a protein, i.e, in this case, how arginine directs the conformational flexibility of the protein and preserves its native folded form. We identified that the exclusion of arginine from the protein surface increases the local structuration of water around the protein, thereby preserving its "biological water" layer, and makes the protein more hydrated at 2 M concentration as compared to the other arginine solutions. Additionally, our microscopic investigation on the interactions of the protein-solvation layer revealed that the structural heterogeneity of the protein surface, arising from the differential physicochemical nature of the amino acid residues, controls the favorable formation of sluggish water-arginine mixed solvation layer at higher arginine concentration that helps the protein to maintain its structural rigidity. Importantly, apart from the protein-solvent hydrogen-bonding interactions, the anion-pi interactions, established between the carboxyl group of arginine and the aromatic amino acid residues of insulin, were recognized to facilitate the protein to maintain its native folded form at the experimental temperatures.

An insight into the binding of 6-hydroxyflavone with hen egg white lysozyme: a combined approach of multi-spectroscopic and computational studies.

The interaction of 6-hydroxyflavone (6HF) with hen egg white lysozyme (HEWL) has been executed using multi-spectroscopic and computational methods. Steady state fluorescence studies indicated that static quenching mechanism is involved in the binding of 6HF with HEWL, which was further supported by excited state lifetime and UV-vis absorption studies. The binding constant (Kb) of the HEWL-6HF complex was observed to be 6.44 ± 0.09 × 104 M-1 at 293 K, which decreases with the increase in temperature. The calculation of the thermodynamic quantities showed that the binding is exothermic in nature with a negative enthalpy change (ΔH = -11.91 ± 1.02 kJ mol-1) along with a positive entropy change (ΔS = +51.36 ± 2.43 J K-1 mol-1), and the major forces responsible for the binding are hydrogen bonding and hydrophobic interactions. The possibility of energy transfer from tryptophan (Trp) residue to the 6HF ligand was observed from Fo¨rster's theory. The inclusion of 6HF within the binding site of HEWL induces some micro-environmental changes around the Trp residues as indicated by synchronous and three-dimensional (3D) fluorescence studies. The changes in secondary structural components of HEWL are observed on binding with 6HF along with a reduction in % α-helical content. Computational studies correlate well with the experimental finding, and the ligand 6HF is found to bind near to Trp 62 and Trp 63 residues of HEWL. Altogether, the present study provides an insight into the interaction dynamics and energetics of the binding of 6HF to HEWL. Communicated by Ramaswamy H. Sarma.

Picosecond Solvation Dynamics in Nanoconfinement: Role of Water and Host-Guest Complexation.

The dynamics of solvation of an excited chromophore, 5-(4″-dimethylaminophenyl)-2-(4'-sulfophenyl)oxazole, sodium salt (DMO), has been explored in confined nanoscopic environments of ß-cyclodextrin (ßCD) and heptakis(2,6-di- O-methyl)-ß-cyclodextrin (DIMEB). Solvation occurs on a distinctly slower time scale (τS3 ∼ 47 ps, τS4 ∼ 517 ps) in the host cavity of DIMEB than in that of ßCD (τS3 ∼ 20 ps, τS4 ∼ 174 ps). The calculated equilibrium solvation response of DMO was characterized by four relaxation components (τS1 ∼ 0.46-0.48 ps, τS2 ∼ 3.2-3.4 ps, τS3 ∼ 32.3-37.7 ps, and τS4 ∼ 232-485 ps), of which the longer ones (τS3, τS4) are well-consistent with experiments, whereas the ultrafast components (τS1, τS2) are unresolved. The observed time constant (τS3) within the ∼20-47 ps range arises from slow water molecules in the primary hydration layers of the host CDs and is slower for DIMEB than for ßCD presumably due to longer-lived and stronger hydrogen bonds that water forms with the former host relative to the latter. Decomposition of the calculated solvation response of DMO has revealed that conformational fluctuations of the macrocyclic hosts give rise to the observed long-time relaxation component (τS4), which is much slower for the inclusion complexes with DIMEB than for those with ßCD because of slower conformational dynamics of the former host than that of the latter.

Conformational disorder and solvation properties of the key-residues of a protein in water-ethanol mixed solutions.

A small number of key-residues in a protein sequence play vital roles in the function, stability, and folding of the protein. The nonuniform conformational disorder of a small protein Chymotrypsin Inhibitor 2 (CI2) and its secondary segments has been quantified in the ethanol governed temperature induced unfolding process by estimating its change in configurational entropy in several water-ethanol mixed solutions. Such calculations further assist us in identifying the key-residues, from where the unfolding of the protein was initiated. Our findings match well with the reported experimental results. We then make an attempt to explore the properties of the solvent water and ethanol around the key-residues of the protein in its folded and unfolded forms at ambient temperature to identify the individual role of ethanol and water in the protein unfolding. We find that the key-residues of the unfolded protein are in good contact with both water and ethanol as compared to those of the folded protein. In the presence of ethanol, water molecules are noticed to form a rigid structurally bound solvation layer around the key-residues of the protein, irrespective of its conformational state. The restricted translational motion and prominent caging effect of the water and ethanol molecules present around the key-residues of the unfolded protein are a signature of the existence of a rigid mixed water-ethanol layer as compared to that around the folded protein. Furthermore, comparable restricted structural relaxation of the key-residue-water and key-residue-ethanol hydrogen bonds in the unfolded protein as compared to that in the folded one implies that the formation of a strong long-lived hydrogen bonding environment nourishes the unfolding process. We believe that our findings will shed light to several co-solvent governed unfolding processes of a protein in general.

Residue Specific Interaction of an Unfolded Protein with Solvents in Mixed Water-Ethanol Solutions: A Combined Molecular Dynamics and ONIOM Study.

The molecular mechanism of ethanol governed unfolding of an enzymatic protein, chymotrypsin inhibitor 2 (CI2), in water-ethanol mixed solutions has been studied by using combined molecular dynamics simulations and ONIOM study. The residue specific solvation of the unfolded protein and the interactions between the individual amino acid residues of the protein with ethanol as well as water have been investigated. The results are compared with that obtained from the folded state of the protein. Further, emphasis has been given to explore the residue's preferential site of attraction toward the nature of the solvents. The heterogeneous structuring of water and ethanol around the hydrophobic and hydrophilic surfaces of the protein is found to correlate well with their available surface areas to the solvents. Both hydrophobic and hydrophilic interactions are found to have important contributions in rupturing protein's secondary structural segments. Further, residue-water as well as residue-ethanol binding energies show significant involvement of the hydrogen bonding environment in the unfolding process; particularly, residue-water hydrogen bonds are found to play an indispensable role.

Drug susceptibility profile of Mycobacterium tuberculosis isolated from HIV infected and uninfected pulmonary tuberculosis patients in eastern India.

HIV is driving the tuberculosis (TB) epidemic in many developing countries including India. This study was initiated to determine the drug resistance pattern of pulmonary TB among 200 HIV seropositive and 50 HIV negative hospitalized patients from different states of Eastern India. The TB positive isolates (120) were screened and characterized by conventional laboratory methods followed by first- and second-line drug susceptibility testing on Lowenstein-Jensen medium by the proportion method. The drug susceptibility testing showed 17.7% (16/90) and 6.6% (2/30) multidrug-resistant (MDR) TB for the HIV positive and HIV negative patients, respectively. 22.2% (4/18) of the isolated MDR-TB cases could be classified as extensively drug-resistant (XDR) TB isolates. 88.8% (16/18) of all the MDR-TB isolates and all XDR-TB isolates were screened from HIV patients. Five (27.7%) of the 18 MDR-TB isolates showed resistance to all the first-line drugs. Mortality rate among the XDR-TB isolates was as high as 75% (3/4). Patients with interrupted anti-TB drug treatment were the ones most affected. These findings are critical and the risk to public health is high, particularly with HIV infected patients.

Current trends of opportunistic infections among HIV-seropositive patients from Eastern India.

In this report we describe the clinical and laboratory profiles of different opportunistic infections (OIs) among 125 immunocompromised patients admitted to a referral hospital in the eastern part of India. Different pathogens were isolated, identified and characterized using the laboratory gold standard methods. Oral candidiasis (88%) was found to be the most common OI, followed by tuberculosis (57%), enteropathogenic Vibrio (47%), cytomegalovirus infection (45%), cryptosporidial diarrhea (43%), Escherichia coli infection (42%) and other infections among the study subjects. Statistical analysis of the case studies shows 120/cumm median CD4+ blood cell count, and the OIs showed an inversely proportional occurrence to the CD4+ count of the immunocompromised patients. The spectrum and frequency of certain OIs highlight the urgency of studying HIV/AIDS in resource-limited countries where locally specific disease patterns may be observed. The purpose of the present investigation was the identification of such opportunistic pathogens, as we feel the HIV epidemic can be more effectively managed if physicians and health planners are aware of this information.