Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.

We demonstrate in this work that the surface tension, water-organic solvent, transfer-free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well-known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25 degrees C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.     

Physical chemical studies of short-chain lecithin homologues. I. Influence of the chain length of the fatty acid ester and of electrolytes on the critical micelle concentration

The critical micelle concentration (CMC) of four synthetic phosphatidylcholines (containing two hexanoyl, heptanoyl, octanoyl or nonanoyl residues respectively) in aqueous solutions have been determined by surface tension measurements. The dependence of the CMC on the chain length is discussed on the basis of the mass action model for micelle formation. For the three higher homologues a contribution of 1.08 kT per CH₂ group to the standard free energy of micellisation is found. The change in this free energy in going from the dihexanoyl- to the diheptanoyllecithin is somewhat larger (1.2 kT per CH₂ group).The influence of high concentrations (several moles per liter) of simple electrolytes on the CMC is interpreted as a salting-out of nonpolar solutes in water. Contrary to expectations the effects of NaCl and Lil on the CMC of dioctanoyllecithin are not additive.     

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 degrees C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E(a) and the free energy of activation DeltaG(0)*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.     

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Measuring enzyme hydration in nonpolar organic solvents using NMR

A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for alpha-chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. (c) 1995 John Wiley & Sons, Inc.     

Molecular dynamic simulations of the water absorbency of hydrogels

A polymer gel can imbibe solvent molecules through surface tension effect. When the solvent happens to be water, the gel can swell to a large extent and forms an aggregate called hydrogel. The large deformation caused by such swelling makes it difficult to study the behaviors of hydrogels. Currently, few molecular dynamic simulation works have been reported on the water absorbing mechanism of hydrogels. In this paper, we first use molecular dynamic simulation to study the water absorbing mechanism of hydrogels and propose a hydrogel-water interface model to study the water absorbency of the hydrogel surface. Also, the saturated water content and volume expansion rate of the hydrogel are investigated by building a hydrogel model with different cross-linking degree and by comparing the water absorption curves under different temperatures. The sample hydrogel model used consists of Polyethylene glycol diglycidyl ether (PEGDGE) as epoxy and the Jeffamine, poly-oxy-alkylene-amines, as curing agent. The conclusions obtained are useful for further investigation on PEGDGE/Jeffamine hydrogel. Moreover, the simulation methods, including hydrogel-water interface modeling, we first propose are also suitable to study the water absorbing mechanism of other hydrogels.     

Conformational changes of peptides at solid/liquid interfaces: a Monte Carlo study

Monte Carlo simulations were performed to study the conformational changes of negatively charged model peptides dissolved in water adsorbed onto charged surfaces. 8-, 16-, and 20-residues peptides were used, each of them consisted of repeating diblock units of aspartic acid (ASP, polar amino acid) and isoleucine (ILE, nonpolar amino acid) residues. We found that a water patch was retained at the charged surface, separating the peptide from it. We believed that these water molecules were primarily responsible for giving a particular orientation to the peptide at the surface. Water did play a role to some extent in the structural stability of the 8-residues peptide. However, for higher chain lengths (16-residues and 20-residues), the intrinsic hydrogen-bonding network (or intrinsic structural stability) showed a predominant effect over hydrophobic dehydration for the stability of the peptide at the surface.     

Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study.

The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are discussed.     

Water and ion permeation in bAQP1 and GlpF channels: a kinetic Monte Carlo study

The kinetic Monte Carlo reaction-path-following technique is applied to determine the lowest-energy water pathway and the coordinating amino acids in bAQP1 and GlpF channels, both treated as rigid. In bAQP1, water molecules pass through the pore between the asparagine-proline-alanine (NPA) and selectivity filter (SF) sites one at a time. The water chain is interrupted at the SF where one water forms three stable hydrogen bonds with protein atoms. In this SF, water's conformation depends on the protonation locus of H182. In GlpF, two water molecules bond simultaneously to the NPA asparagines and pass through the SF in zigzag fashion. No water single-file forms in rigid GlpF. To accommodate a single file of waters requires narrowing the GlpF pore. Our results reveal that in both proteins a proposed bipolar water arrangement is thermally disrupted in the NPA region, especially in the cytoplasmic part of the pore. The equilibrium hydrogen-bonded chain is occasionally interrupted in the hydrophobic zones adjacent to the NPA motifs. The permeation of alkali cations through bAQP1 and GlpF is barred due to a large free-energy barrier in the NPA region as well as a large energy barrier blocking entry from the cytoplasm. Permeation of halides is prevented due to two large energy barriers in the cytoplasmic and periplasmic pores as well as a large free-energy barrier barring entry from the periplasm. Our results, based on modeling charge permeation, support an electrostatic rather than orientational basis for proton exclusion. Binding within the aquaporin pore cannot compensate sufficiently for dehydration of the protonic charge; there is also an electrostatic barrier in the NPA region blocking proton transport. The highly ordered single file of waters, which is drastically interrupted at the SF of bAQP1, may also contribute to proton block.     

Origins of protein denatured state compactness and hydrophobic clustering in aqueous urea: inferences from nonpolar potentials of mean force

Free energies of pairwise hydrophobic association are simulated in aqueous solutions of urea at concentrations ranging from 0-8 M. Consistent with the expectation that hydrophobic interactions are weakened by urea, the association of relatively large nonpolar solutes is destabilized by urea. However, the association of two small methane-sized nonpolar solutes in water has the opposite tendency of being slightly strengthened by the addition of urea. Such size effects and the dependence of urea-induced stability changes on the configuration of nonpolar solutes are not predicted by solvent accessible surface area approaches based on energetic parameters derived from bulk-phase solubilities of model compounds. Thus, to understand hydrophobic interactions in proteins, it is not sufficient to rely solely on transfer experiment data that effectively characterize a single nonpolar solute in an aqueous environment but not the solvent-mediated interactions among two or more nonpolar solutes. We find that the m-values for the rate of change of two-methane association free energy with respect to urea concentration is a dramatically nonmonotonic function of the spatial separation between the two methanes, with a distance-dependent profile similar to the corresponding two-methane heat capacity of association in pure water. Our results rationalize the persistence of residual hydrophobic contacts in some proteins at high urea concentrations and explain why the heat capacity signature (DeltaC(P)) of a compact denatured state can be similar to DeltaC(P) values calculated by assuming an open random-coil-like unfolded state.     

Physicochemical properties of structured water in human albumin and gammaglobulin solutions.

The physicochemical properties of the different phases of water molecules were studied in concentrated solutions (132 g/L) of human serum albumin and gammaglobulin by (1)H NMR relaxometry. Spin-lattice (T1) relaxation times of total water and structured water (non-freezable water) were measured at 40 MHz above and below the freezing point of bulk water (ordinary, liquid water) at different temperatures. Analysis of the temperature dependence of the T1 demonstrated that total water differed qualitatively while structured water characteristics changed both quantitatively and qualitatively in the two protein solutions. Comparison of the temperature dependence of the structured water's T1 in the two solutions in the presence of an increasing concentration of manganese chloride allowed two main conclusions to be drawn. Firstly, the differences observed in total water and structured water physicochemical properties are directly related to protein structure and three-dimensional arrangement. Secondly, the motion of structured water determines the motion of the total water in the system through the values of the translational diffusion and chemical exchange correlation times tau(D) and tau m.     

On the calculation of binding free energies using continuum methods: Application to MHC class I protein-peptide interactions

This paper describes a methodology to calculate the binding free energy (ΔG) of a protein-ligand complex using a continuum model of the solvent. A formal thermodynamic cycle is used to decompose the binding free energy into electrostatic and non-electrostatic contributions. In this cycle, the reactants are discharged in water, associated as purely nonpolar entities, and the final complex is then recharged. The total electrostatic free energies of the protein, the ligand, and the complex in water are calculated with the finite difference Poisson-Boltzmann (FDPB) method. The nonpolar (hydrophobic) binding free energy is calculated using a free energy-surface area relationship, with a single alkane/water surface tension coefficient (γ_aw). The loss in backbone and side-chain configurational entropy upon binding is estimated and added to the electrostatic and the nonpolar components of ΔG. The methodology is applied to the binding of the murine MHC class I protein H-2K^b with three distinct peptides, and to the human MHC class I protein HLA-A2 in complex with five different peptides. Despite significant differences in the amino acid sequences of the different peptides, the experimental binding free energy differences (ΔΔG_exp) are quite small (<0.3 and <2.7 kcal/mol for the H-2K^b and HLA-A2 complexes, respectively). For each protein, the calculations are successful in reproducing a fairly small range of values for ΔΔG_calc (<4.4 and <5.2 kcal/mol, respectively) although the relative peptide binding affinities of H-2K^b and HLA-A2 are not reproduced. For all protein-peptide complexes that were treated, it was found that electrostatic interactions oppose binding whereas nonpolar interactions drive complex formation. The two types of interactions appear to be correlated in that larger nonpolar contributions to binding are generally opposed by increased electrostatic contributions favoring dissociation. The factors that drive the binding of peptides to MHC proteins are discussed in light of our results.     

Solvent interactions with the Trp-cage peptide in 35% ethanol-water

Intermolecular NOE experiments have been used to explore interactions of water and ethanol molecules in 35% ethanol/65% water (v/v) with the peptide Trp-cage at temperatures from 5 to 25 degrees C. Magnetic dipole-dipole cross-relaxation terms sigma(HH) (NOE) and sigma(HH) (ROE) for interaction of solvent components with spins of the peptide suggest that ethanol molecules associate with backbone atoms for times of the order of nanoseconds at 5 degrees C. Formation of peptide-ethanol complexes can also account for the larger-than-expected values of cross-relaxation terms at higher temperatures. Hydrocarbon side chains of the peptide do not appear to experience such interactions with ethanol. Cross relaxation resulting from water-peptide interactions are consistent with long-lived water interactions with the backbone atoms. Water cross relaxation with nonpolar side chains of the peptide (Leu2, Ile4, Leu7, and proline residues) are only those expected for bulk solvent. However, long-lived association of both water and ethanol with the polar side chains of Tyr3 and Trp6 is indicated by the data. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 862-872, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 °C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E_a and the free energy of activation ΔG₀*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.     

Combined monte carlo and molecular dynamics simulation of fully hydrated dioleyl and palmitoyl-oleyl phosphatidylcholine lipid bilayers

We have applied a new equilibration procedure for the atomic level simulation of a hydrated lipid bilayer to hydrated bilayers of dioleyl-phosphatidylcholine (DOPC) and palmitoyl-oleyl phosphatidylcholine (POPC). The procedure consists of alternating molecular dynamics trajectory calculations in a constant surface tension and temperature ensemble with configurational bias Monte Carlo moves to different regions of the configuration space of the bilayer in a constant volume and temperature ensemble. The procedure is applied to bilayers of 128 molecules of POPC with 4628 water molecules, and 128 molecules of DOPC with 4825 water molecules. Progress toward equilibration is almost three times as fast in central processing unit (CPU) time compared with a purely molecular dynamics (MD) simulation. Equilibration is complete, as judged by the lack of energy drift in 200-ps runs of continuous MD. After the equilibrium state was reached, as determined by agreement between the simulation volume per lipid molecule with experiment, continuous MD was run in an ensemble in which the lateral area was restrained to fluctuate about a mean value and a pressure of 1 atm applied normal to the bilayer surface. Three separate continuous MD runs, 200 ps in duration each, separated by 10,000 CBMC steps, were carried out for each system. Properties of the systems were calculated and averaged over the three separate runs. Results of the simulations are presented and compared with experimental data and with other recent simulations of POPC and DOPC. Analysis of the hydration environment in the headgroups supports a mechanism by which unsaturation contributes to reduced transition temperatures. In this view, the relatively horizontal orientation of the unsaturated bond increases the area per lipid, resulting in increased water penetration between the headgroups. As a result the headgroup-headgroup interactions are attenuated and shielded, and this contributes to the lowered transition temperature.     

Explicit and implicit water simulations of a beta-hairpin peptide.

The conformational properties of a beta-hairpin peptide (YITNSDGTWT) were studied by using both explicit and implicit water simulations. The conformational space of the peptide was scanned by using a restricted hydrogen-bonding search method. The search method used generated the conformational space with enough diversity and good representation of beta-hairpin structures. By using a total surface area-based treatment of hydrophobic interactions, implicit water simulations failed to discriminate between experimental beta-hairpin structures from the rest of the conformers present in the authors' conformation library. However, with inclusion of vibrational free energy and accounting separately for polar and nonpolar surface areas, the nuclear magnetic resonance structure was ranked successfully as the most stable conformation. There is a loose correlation between the conformational energies by the continuum model and the conformational energies by explicit water simulation for conformers with similar structures. However, in terms of solvation energy, both approaches have a much better correlation. By using proper treatment of surface effect (partition of the surface area into polar and nonpolar areas) and including vibrational free-energy contribution, the continuum models should be reliable. Furthermore, the authors found that, for this peptide, beta-hairpin structures have large vibrational entropy that contributes decisively to the stability of folded beta-hairpin structures. Proteins 1999;37:73-87.     

Inspecting fluctuation and coordination around chromophore inside green fluorescent protein from water to nonpolar solvent

Green fluorescent protein (GFP) is a widely used biomarker that demands systematical rational approaches to its structure function redesign. In this work, we mainly utilized atomistic molecular dynamics simulations to inspect and visualize internal fluctuation and coordination around chromophore inside GFP, from water to nonpolar octane solvent. We found that GFP not only maintains its β-barrel structure well into the octane, but also sustains internal residue and water coordination to position the chromophore stably while suppress dihedral fluctuations of the chromophore, so that functional robustness of GFP is achieved. Our accompanied fluorescence microscope measurements accordingly confirmed the GFP functioning into the octane. Furthermore, we identified that crucial water sites inside GFP along with permeable pores on the β-barrel of the protein are largely preserved from the water to the octane solvent, which allows sufficiently fast exchanges of internal water with the bulk or with the water layer kept on the surface of the protein. By additionally pulling GFP from bulk water to octane, we suggest that the GFP function can be well maintained into the nonpolar solvent as long as, first, the protein does not denature in the nonpolar solvent nor across the polar-nonpolar solvent interface; second, a minimal set of water molecules are in accompany with the protein; third, the nonpolar solvent molecules may need to be large enough to be nonpermeable via the water pores on the β-barrel.     

Effect of oligosaccharides and their monosaccharide mixtures on the stability of proteins: a scaled particle study

Experimental results of RNase-A stabilization by sugar osmolytes show that the change in the Gibbs free energy (ΔGD) associated with the equilibrium, N (native) state ? D (denatured) state of the protein in the presence of equimolar mixture of monosaccharides is larger than that of the corresponding oligosaccharides at a given temperature and pH. However, at the molar scale, ΔGD obtained in the presence of an oligosaccharide is much higher as compared with ΔGD obtained using individual monosaccharide. We used scaled particle theory (SPT) to explain these experimental observations. The effective length, called Tolman's length that describes the curvature correlations to a surface area or surface tension and in turn contributes to the change in free energy, is discussed. Tolman's length is higher for corresponding monomer mixture than the oligosaccharide molecules. Based on SPT analysis, a geometrical model is proposed for clustering of monosaccharides in the mixture due to high particle density. The cluster is presumed to have weak interaction among them due to larger hydrodynamic radius than that of the bonded molecules of oligosaccharides.     

Molecular dynamics simulation of solvated protein at high pressure.

We have completed a molecular dynamics simulation of protein (bovine pancreatic trypsin inhibitor, BPTI) in solution at high pressure (10 kbar). The structural and energetic effects of the application of high pressure to solvated protein are analyzed by comparing the results of the high-pressure simulation with a corresponding simulation at low pressure. The volume of the simulation cell containing one protein molecule plus 2943 water molecules decreases by 24.7% at high pressure. This corresponds to a compressibility for the protein solution of beta = 1.8 x 10(-2) kbar-1. The compressibility of the protein is estimated to be about one-tenth that of bulk water, while the protein hydration layer water is found to have a greater compressibility as compared to the bulk, especially for water associated with hydrophobic groups. The radius of gyration of BPTI decreases by 2% and there is a one third decrease in the protein backbone atomic fluctuations at high pressure. We have analyzed pressure effects on the hydration energy of the protein. The total hydration energy is slightly (4%) more favorable at high pressure even though the surface accessibility of the protein has decreased by a corresponding amount. Large pressure-induced changes in the structure of the hydration shell are observed. Overall, the solvation shell waters appear more ordered at high pressure; the pressure-induced ordering is greatest for nonpolar surface groups. We do not observe evidence of pressure-induced unfolding of the protein over the 100-ps duration of the high-pressure simulation. This is consistent with the results of high-pressure optical experiments on BPTI.(ABSTRACT TRUNCATED AT 250 WORDS)