The role of hydrophobicity in the cold denaturation of proteins under high pressure: A study on apomyoglobin.

An exciting debate arises when microscopic mechanisms involved in the denaturation of proteins at high pressures are explained. In particular, the issue emerges when the hydrophobic effect is invoked, given that hydrophobicity cannot elucidate by itself the volume changes measured during protein unfolding. In this work, we study by the use of molecular dynamics simulations and essential dynamics analysis the relation between the solvation dynamics, volume, and water structure when apomyoglobin is subjected to a hydrostatic pressure regime. Accordingly, the mechanism of cold denaturation of proteins under high-pressure can be related to the disruption of the hydrogen-bond network of water favoring the coexistence of two states, low-density and high-density water, which directly implies in the formation of a molten globule once the threshold of 200 MPa has been overcome.

Pressure effect on micellization of non-ionic surfactant Triton X-100.

Micellar aggregates can be arranged in new types of conformational assemblies when they are isotropically compressed. Thus, the pressure effects in the underlying fundamental interactions leading to self-assembly of micellar aggregates can be represented by changes in the phase boundaries with increasing pressure. In this paper, we have employed molecular dynamics simulations to study the self-assembly of micelles composed of the non-ionic surfactant Triton X-100 at the atomic scale, monitoring the changes in the solvation dynamics when the micelles are subjected to a wide range of hydrostatic pressures. The computational molecular model was capable of self-assembling and forming a non-ionic micelle, which subsequently was coupled to a high-pressure barostat producing a geometric transition of the micelle due to changes in the solvation dynamics. Accordingly, under a high pressure regime, the hydrogen bonds are redistributed, the water density is modified, and water acts as an unstructured liquid, capable of penetrating into the micelle.

Essential dynamics of the cold denaturation: pressure and temperature effects in yeast frataxin.

The cold denaturation of globular proteins is a process that can be caused by increasing pressure or decreasing the temperature. Currently, the action mechanism of this process has not been clearly understood, raising an interesting debate on the matter. We have studied the process of cold denaturation using molecular dynamics simulations of the frataxin system Yfh1, which has a dynamic experimental characterization of unfolding at low and high temperatures. The frataxin model here studied allows a comparative analysis using experimental data. Furthermore, we monitored the cold denaturation process of frataxin and also investigated the effect under the high-pressure regime. For a better understanding of the dynamics and structural properties of the cold denaturation, we also analyzed the MD trajectories using essentials dynamic. The results indicate that changes in the structure of water by the effect of pressure and low temperatures destabilize the hydrophobic interaction modifying the solvation and the system volume leading to protein denaturation. Proteins 2016; 85:125-136. © 2016 Wiley Periodicals, Inc.

Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal Epithelial Cells.

Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-ß), or pro-inflammatory mediators (IL-1ß and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489-2496, 2017. © 2016 Wiley Periodicals, Inc.

Molecular dynamics of water in the neighborhood of aquaporins.

Present knowledge obtained by molecular dynamics (MD) simulation studies regarding the dynamics of water, both in the vicinity of biological membranes and within the proteinaceous water channels, also known as aquaporins (AQPs), is reviewed. A brief general summary of the water models most extensively employed in MD simulations (SPC, SPC/E, TIP3P, TIP4P), indicating their most relevant pros and cons, is likewise provided. Structural considerations of water are also discussed, based on different order parameters, which can be extracted from MD simulations as well as from experiments. Secondly, the behaviour of water in the neighbourhood of membranes by means of molecular dynamics simulations is addressed. Consequently, the comparison with previous experimental evidence is pointed out. In living cells, water is transported across the plasma membrane through the lipid bilayer and the aforementioned AQPs, which motivates this review to focus mostly on MD simulation studies of water within AQPs. Relevant contributions explaining peculiar properties of these channels are discussed, such as selectivity and gating. Water models used in these studies are also summarised. Finally, based on the information presented here, further MD studies are encouraged.

Aggregation of non-polar solutes in water at different pressures and temperatures: the role of hydrophobic interaction.

Due to the importance of the hydrophobic interaction in protein folding, we decided to study the effect of pressure and temperature on the phase transitions of non-polar solutes in water, and thereby their solubility, using molecular dynamics simulations. The main results are: (1) within a certain range, temperature induces the aggregation of Lennard-Jones particles in water; and (2) pressure induces disaggregation of the formed clusters. From the simulated data, a non-monotonic coexistence curve for the binary system was obtained, from which a critical point of T(c) = 383 ± 9 K and p(c) = 937 ± 11 bar was determined. The results are in accordance with previous experimental evidence involving transitions of hydrocarbons in water mixtures, and protein unfolding.

The behavior of the hydrophobic effect under pressure and protein denaturation.

It is well known that proteins denature under high pressure. The mechanism that underlies such a process is still not clearly understood, however, giving way to controversial interpretations. Using molecular dynamics simulation on systems that may be regarded experimentally as limiting examples of the effect of high pressure on globular proteins, such as lysozyme and apomyoglobin, we have effectively reproduced such similarities and differences in behavior as are interpreted from experiment. From the analysis of such data, we explain the experimental evidence at hand through the effect of pressure on the change of water structure, and hence the weakening of the hydrophobic effect that is known to be the main driving force in protein folding.

Prediction of the most favorable configuration in the ACBP-membrane interaction based on electrostatic calculations.

Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP-membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding (dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP-membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.

A hydrophobic loop in acyl-CoA binding protein is functionally important for binding to palmitoyl-coenzyme A: a molecular dynamics study.

Acyl-CoA binding protein (ACBP) plays a key role in lipid metabolism, interacting via a partly unknown mechanism with high affinity with long chain fatty acyl-CoAs (LCFA-CoAs). At present there is no study of the microscopic way ligand binding is accomplished. We analyzed this process by molecular dynamics (MDs) simulations. We proposed a computational model of ligand, able to reproduce some evidence from nuclear magnetic resonance (NMR) data, quantitative time resolved fluorometry and X-ray crystallography. We found that a hydrophobic loop, not in the active site, is important for function. Besides, multiple sequence alignment shows hydrophobicity (and not the residues itselves) conservation.

Pressure denaturation of apomyoglobin: a molecular dynamics simulation study.

The effect of pressure on the structure and mobility of Sperm Wale Apomyoglobin was studied by Molecular Dynamics computer simulation at 1 bar and 3 kbar (1 atm=1.01325 bar=101.325 kPa). The results are in good agreement with the available experimental data, allowing further analysis of other features of the effect of pressure on the protein solution. From the analysis of Secondary Structures (SS) along the trajectories it is observed that alpha-helixes are favoured under pressure at the expense of bends, turns and 3-helixes. The studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. The studies of tertiary structure show important conformational changes. The evolution of the Solvent Accessed Surface (SAS) with pressure shows a notorious increase due almost completely to a biased raise in the hydrophobic area exposed, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions.

Effect of pressure on the conformation of proteins. A molecular dynamics simulation of lysozyme.

The effect of pressure on the structure and mobility of lysozyme was studied by molecular dynamics computer simulation at 1 and 3 kbar (1 atm = 1.01325 bar = 101.325 kPa). The results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. The studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. From the analysis of secondary structure along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. The difference in solvent-accessed surface (SAS) with pressure shows a clear inversion of the hydrophilic/hydrophobic SAS ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions.

Free energy perturbation calculations on glucosidase-inhibitor complexes.

Free energy perturbation studies have been performed on Glucoamylase II (471) from Aspergillus awamori var. X100 complexed with three different inhibitors: (+)lentiginosine, (+)(1S,2S,7R,8aS) 1,2,7-trihydroxyindolizidine, (+)(1S,2S,7S,8aS) 1,2,7-trihydroxyindolizidine and the inactive compound (+)(1S,7R,8aS)-1,7-dihydroxyindolizidine. Molecular dynamic simulations were carried out using a recently developed procedure for fast Free Energy Perturbation calculations. In this procedure only a sphere of 1.8 nm around the central atom of the inhibitor is considered in the calculations. Crystallographic restraints are applied over this reduced system using a generated electron density map. The obtained values for the free energy differences agree with experimental data showing the importance of fast calculations in drug design even when the crystallographic structure of the complex is not available. As the method uses only the crystallographic structure of the receptor, it is possible to test the possible efficiency of even still not synthesised ligands, making the pre-selection of compounds much easy and faster.

Molecular dynamics simulation for ligand-receptor studies. Carbohydrates interactions in aqueous solutions.

The review deals with the problem of the study of ligand-receptor interactions and the use of Molecular Dynamics (MD) simulation to approach such a problem. After a short review of the fundamentals of MD we describe the medium in which all biology takes place, water. Emphasis is put on the water models appropriate for simulation of macromolecular systems explicitly including the water molecules. We consider the quality of the water model both in terms of simplicity and performance to describe the liquid water properties. Heavy water, although not a biologically viable medium, is considered since many experiments make use of it as a solvent. Sweetness of carbohydrates is considered as an example of the procedure suitable to characterize active sites on the ligands. Consideration is given to the computation of the binding constants through molecular dynamics. The computation of the Free Energy is described and illustrated. The potentiality of MD for studies of ligand-receptor interactions is limited by the computer resources, for even with large computing facilities the need of relatively long simulation times severely restricts the study of large systems. A method is described in which several shells are treated at different levels of approximation, form mechanical response and mean electrical field to quantum mechanics, through stochastic dynamics and atomic classical MD. The review closes with a brief account of the perspectives of the method.

Collagen stability, hydration and native state.

Molecular dynamics simulations of a collagen-like peptide (Pro-Hyp-Gly)4-Pro-Hyp-Ala-(Pro-Hyp-Gly)5 have been done in order to study the contribution of the hydration structure on keeping the native structure of collagen. The simulation shows that the absence of water produces a distortion on the molecular conformation and an increase in the number of intra-molecular hydrogen bonds. This is in agreement with previous experimental results showing the stiffness of collagen under severe drying and its increase in the thermal stability. This dehydrated material does not keep, however, the native structure.

Stochastic molecular dynamics of peanut lectin PNA complex with T-antigen disaccharide.

The stochastic boundary molecular dynamics simulation method was applied to investigate the structure of a complex comprised of a tetrameric peanut lectin and the tumour-associated disaccharide, Galbeta1-3GalNAc (T-antigen). Only a small region encompassing the active site was explicitly included in the calculations, but the electrical contribution of most outer atoms was taken into account by adding to the effective potential a term coming from an electrostatic potential grid that was pre-calculated and used to approximate the electrostatic energy and the force at any point in the interacting site. Results of simulating the intermolecular hydrogen bond network agree fairly well with X-ray experiments. An estimation of the direct and water-mediated interaction mean lifetimes and mean water residence times around the T-antigen oxygen atoms was computed over 400 ps. Monitoring the behaviour of water molecules within the active site revealed that there is a constant exchange of water with the bulk, especially in the proximity of ASN41, ASN127 and GLU129. The temporal evolution of the glycosidic linkage was also investigated and compared with simulations of T-antigen in solution. These studies of peanut lectins-sugar complexes clearly emphasize the importance of bound water molecules in generating carbohydrate specificity.

A molecular dynamics approach to ligand-receptor interaction in the aspirin-human serum albumin complex.

In this work, we present a study of the interaction between human serum albumin (HSA) and acetylsalicylic acid (ASA, C(9)H(8)O(4)) by molecular dynamics simulations (MD). Starting from an experimentally resolved structure of the complex, we performed the extraction of the ligand by means of the application of an external force. After stabilization of the system, we quantified the force used to remove the ASA from its specific site of binding to HSA and calculated the mechanical nonequilibrium external work done during this process. We obtain a reasonable value for the upper boundary of the Gibbs free energy difference (an equilibrium thermodynamic potential) between the complexed and noncomplexed states. To achieve this goal, we used the finite sampling estimator of the average work, calculated from the Jarzynski Equality. To evaluate the effect of the solvent, we calculated the so-called "viscous work," that is, the work done to move the aspirin in the same trajectory through the solvent in absence of the protein, so as to assess the relevance of its contribution to the total work. The results are in good agreement with the available experimental data for the albumin affinity constant for aspirin, obtained through quenching fluorescence methods.

Islet neogenesis associated protein (ingap): structural and dynamical properties of its active pentadecapeptide.

We have studied the structural and dynamical properties of the biologically active pentadecapeptide of the islet neogenesis associated protein (INGAP-PP) and of two other pentadecapeptides with the same amino acid composition but randomly scrambled primary sequences, using molecular dynamic simulations. Our data demonstrates that whilst the peptides with scrambled sequences show no definite prevalent structure in solution, INGAP-PP maintains a notably stable tertiary fold, namely, a conformer with a central beta-sheet and closed C-terminal. Such structure resembles the one corresponding to the amino acid sequence of human pancreatitis associated protein-1 (PAP-1), which presents 85% sequence homology with INGAP. These results could reasonably explain why the two scrambled sequences tested showed no biological activity, while INGAP-PP significantly increases beta-cells function and mass both in vitro and in vivo conditions. The capability of INGAP-PP to temporarily adopt other closely related conformations offers also a plausible explanation for the 50 fold experimental difference in potency between the active pentadecapeptide and the whole protein. They also suggest that the C-terminal region of INGAP-PP may plausibly be the locus for its interaction with the cell receptor. Consequently, the knowledge gathered through our data can help to obtain more potent INGAP-PP analogs, suitable for the prevention and treatment of diabetes.

Clustering of Lennard-Jones particles in water: temperature and pressure effects.

While the hydrophobic effect is, for many systems, one of the most relevant interactions, it may be said that in the case of biological systems this effect becomes of determinant importance. Although the matter has been analyzed extensively, certain aspects are yet to be elucidated. Hence, the study on the behavior of the hydrophobic effect with temperature, and particularly with pressure deserves further investigation; model systems may help us in the task. We have analyzed the behavior of Lennard-Jones particles in water by means of molecular dynamics simulation under different conditions of size, concentration, temperature, and pressure. Following the formation of particle aggregates we can observe an increase of the hydrophobic effect with temperature and a strong weakening of the effect at high pressures. The results agree with the experimental evidence and show the ability of molecular dynamics simulation to account for the behavior of nonpolar substances under different conditions, provided that the intermolecular interactions used are adequate.

Classical molecular-dynamics simulation of the hydroxyl radical in water.

We have studied the hydration and diffusion of the hydroxyl radical OH0 in water using classical molecular dynamics. We report the atomic radial distribution functions, hydrogen-bond distributions, angular distribution functions, and lifetimes of the hydration structures. The most frequent hydration structure in the OH0 has one water molecule bound to the OH0 oxygen (57% of the time), and one water molecule bound to the OH0 hydrogen (88% of the time). In the hydrogen bonds between the OH0 and the water that surrounds it the OH0 acts mainly as proton donor. These hydrogen bonds take place in a low percentage, indicating little adaptability of the molecule to the structure of the solvent. All hydration structures of the OH0 have shorter lifetimes than those corresponding to the hydration structures of the water molecule. The value of the diffusion coefficient of the OH0 obtained from the simulation was 7.1x10(-9) m2 s(-1), which is higher than those of the water and the OH-.

The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin.

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.