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Amino Acid Side Chain Interactions in the Presence of Salts * Author to whom correspondence should be addressed. Phone: (301) 402-1382. Fax: (301) 402-2867. E-mail: mago/at/helix.nih.gov ![]() ![]() | |||||||||||||||||
Abstract The effects of salt on the intermolecular interactions between polar/charged amino acids are investigated through molecular dynamics simulations. The mean forces and associated potentials are calculated for NaCl salt in the 0–2 M concentration range at 298 K. It is found that the addition of salt may stabilize or destabilize the interactions, depending on the nature of the interacting molecules. The degree of (de)stabilization is quantified, and the origin of the salt-dependent modulation is discussed based upon an analysis of solvent density profiles. To gain insight into the molecular origin of the salt modulation, spatial distribution functions (sdf’s) are calculated, revealing a high degree of solvent structuredness in all cases. The peaks in the sdf’s are consistent with long-range hydrogen-bonding networks connecting the solute hydrophilic groups, and that contribute to their intermolecular solvent-induced forces. The restructuring of water around the solutes as they dissociate from close contact is analyzed. This analysis offers clues on how the solvent structure modulates the effective intermolecular interactions in complex solutes. This modulation results from a critical balance between bulk electrostatic forces and those exerted by (i) the water molecules in the structured region between the monomers, which is disrupted by ions that transiently enter the hydration shells, and (ii) the ions in the hydration shells in direct interactions with the solutes. The implications of these findings in protein/ligand (noncovalent) association/dissociation mechanisms are briefly discussed. | |||||||||||||||||
Biopolymers such as proteins and nucleic acids are the basic components of biological systems. Their dynamics, conformations, and interactions in the cell determine the behavior of living organisms. Many of the physical and chemical properties of these molecules have been studied using experimental and computational techniques. However, important processes underlying biological function, such as protein–ligand interactions, molecular recognition, aggregation, and protein folding, are not yet well understood. Biomolecular interactions, either in vivo or in vitro, occur mainly in aqueous environments that may vary broadly in composition. Solvents not only affect the conformation, dynamics, and thermodynamics of biomolecules1,2 but also modulate their chemical properties.3 In proteins, the solvent controls chemical reactions, e.g., in enzyme catalysis, and modulates noncovalent interactions, thus governing the dynamics of molecular association and dissociation.4–6 The addition of salts and cosolvents to the solution can cause significant changes in many biomolecular properties.7–13 For example, protein solubility can change by the addition of salts to the solution.14 In general, the solubility increases slightly (salting in) at low salt concentrations and drops sharply (salting out) at higher concentrations. Salting out is a common experimental procedure to precipitate proteins and separate them from a solution. The change in protein solubility as well as other properties of proteins in solutions depend on the nature of the salts and cosolvents.15 Progress has been made in understanding the effects of the Hofmeister series on simple solutes,16,17 but a clear understanding of their molecular origins in complex solutes has been more elusive. Changing the conditions of the solution can also affect the thermodynamics of single proteins, altering their structural stability.10,13 Thus, protein denaturation is promoted by changing the concentrations of alcohols, urea, and guanidine hydrochloride, while protein stabilization can be reinforced with the addition of sucrose, certain amino acids, and salts. Because salts and other electrolytes are ubiquitous in biological systems, understanding their effects on proteins behavior at the molecular level is important to quantify their interactions in a biological or experimental context. Because of the complex nature of these interactions, they do not lend themselves readily to theoretical approximations. Therefore, molecular dynamics (MD) simulations can be used to explore the microscopic origin of such interactions. MD simulations have been used to investigate physical and chemical properties of liquids18–20 and to study biomolecular processes at an atomic level of detail.21,22 Bulk electrostatic modulation of molecular interactions originates in the polarization and reorientation of water molecules in the bulk phase. Protein electrostatics is an important component of intermolecular interactions23,24 and may determine protein–ligand association and binding free energies.25,26 Proteins known to interact mainly by electrostatic forces have been engineered to accelerate the rate of association and the formation of tighter molecular complexes.27,28 Besides bulk electrostatics, other solvent-induced forces (SIFs) result from the rearrangement of water molecules around the solute due to their exclusion from the region occupied by the solute itself.29,30 This rearrangement modifies (when compared to bulk liquid) the hydrogen-bonding (HB) network of water around the solute, generating forces and torques that affect its equilibrium structure and dynamics. These forces operate regardless of the polar character of the solute; e.g., hydrophobic forces31–33 are SIFs between nonpolar molecules. Hydrophobic forces make important contributions to protein–ligand binding, are linked to protein recognition and specificity, and play a role in early protein folding events.34,35 For polar and charged solutes36 the rearrangement of the excluded solvent and its HB network is locally perturbed by the electric field. Therefore, the microscopic origin of the SIF is more complex and indirectly affected by the field. In this case the formation of solute–solvent–solute HB may result in so-called hydrophilic forces37 (i.e., SIF between hydrophilic groups) that may also affect protein–ligand interactions and protein folding.4,30,38 Both electrostatics and solvent-induced forces are modified by the presence of salts.25,26,39–41 A recent study42 quantified the extent in which salt concentration strengthens the hydrophobic interaction between two methane molecules. For polar and charged solutes bulk electrostatic forces and SIFs operate simultaneously. A systematic study of salt effects on the intermolecular interactions in these systems has not yet been reported and is presented here. Extensive MD simulations are carried out to calculate the intermolecular mean forces (MF) between amino acid pairs and their associated potentials (PMFs) and to quantify the changes induced by the ion atmosphere at different salt concentrations. The molecular origin of such modulations is investigated. | |||||||||||||||||
Structurally simple solutes (e.g., methane molecules) have proven useful in gaining insight into the salt-dependent modulation at the molecular level. A certain degree of structural complexity is desirable, however, to study biomolecular interactions more realistically. Amino acids differ broadly in their topologies and chemical properties and are simple enough for the systematic study sought herein. Thus, eight amino acid dimers were modeled here as described earlier43 by combining five polar/charged acceptor/donor molecules: Asp− –Arg+, Asp− –Lys+, Asp− –His+, Asp− –Ser, Ser–Arg+, Ser–Lys+, Ser–His+, and Ser–Ser; a dimer is then defined as two monomers (each monomer being a single amino acid) interacting through noncovalent forces. Details of the computational setup were reported in ref 43; an overview and additional details are given below for completeness. The carboxy and amino termini of each amino acid were capped with uncharged groups. Each dimer was immersed in a cubic box of volume ~(46 Å)3, containing TIP3P water molecules at T = 298 K and a density of ρw ≈ 0.993 g/cm3. Salt concentrations of 0.1, 0.5, 1.0, and 2.0 M (mol/L; equal to the ionic strength in this case) are considered; Na+ and Cl− ions were introduced by replacing water molecules (one per ion) randomly; additional ions were added to neutralize the system when required.43 MD simulations were carried out using periodic boundary conditions (PBC) and particle mesh Ewald (PME) summations; the all-atom CHARMM force field was used.44 The system was initially equilibrated for 1 ns to allow ions to diffuse and accommodate around the solutes at their initial configuration (proton–acceptor distance of 1 Å). The convergence of the spatial distribution of ions was not quantified but assessed by visual inspection of their spatial distribution functions (sdf’s; see below). The distance r between the monomers was then increased in successive steps of Δr = 0.2Å along the line connecting the donor, the shared proton, and the acceptor atoms as described;43 an equilibration phase of 100 ps followed each distance update to relax local perturbations of the liquid. The production phase comprised a set of successive simulations of τ = 240 ps each, adding up to a total production time of τT ≈ 15 ns (a 4-fold increase with respect to the simulations in pure water reported previously,43 and needed here to reduce statistical errors for the comparison of the PMF). | |||||||||||||||||
A one-letter code will be used for the amino acids as follows: R = Arg+, K = Lys+, H = His+, D = Asp−, and S = Ser. Figure 1A shows the PMF, VM(r), for the DR dimer at different salt concentrations (indicated by the index M; V0(r) corresponds to pure water); adding salt stabilizes the intermolecular interaction in this case. The inset in Figure 1A shows VM(r) at r = rcm, rts, and rss, corresponding to the contact, transition state (desolvation barrier), and solvent-separated proton–acceptor distances, respectively. At the contact minimum, the dimer stabilizes by ~1 kcal/mol at 1 M and by ~2 kcal/mol at 2 M. PMF plots for the SR dimer are presented in Figure 2A, showing the opposite effect of salt, i.e., neutralizing the acceptor molecule destabilizes the interactions; e.g., at 2 M an increase of ~1 kcal/mol is observed at the contact distance. Adding salt also strengthens the interactions of DS and SS dimers (cf. Figures 3A and 4A). Figure 5 displays the potentials VM(rcm) with respect to pure water, at different salt concentrations (i.e., ΔVM(rcm) = VM(rcm) – V0(rcm)) for the eight dimers studied here; a summary of the calculated values of ΔVM(r) at rcm, rts, and rss is given in Table 1. Figure 5 shows no obvious correlation between the strength of the (de)stabilization and the polar/charged nature of the monomers. Thus, the interactions between charged species may either stabilize (DR) or destabilize (DK) with added salt or show little variations as in DH. A similar lack of correlation is observed for dimers containing one neutral molecule; SR destabilizes by ~1 kcal/mol, while SH stabilizes by the same amount at 2 M; adding salt also stabilizes the SS dimer by more than ~1 kcal/mol at 2 M. These observations suggest that a critical balance of solvent forces may be operating on the solutes in the 0–2 M salt concentration range. It is then of interest to analyze the solvent component, Fs,M(r), of the intermolecular mean forces (MFs) for the four representative dimers discussed above as well as their effects on the potentials. As shown in Figure 5, the larger effects of salts are observed at higher concentrations (~1–2 M), so only the forces and potentials in pure water and in 2 M solution are discussed.
Figure 1B shows the change of the intermolecular potential for the DR dimer in 2 M solution with respect to pure water as a function of the intermolecular distance, i.e., ΔV2(r) = V2(r) – V0(r). As the intermolecular distance decreases, ΔV2(r) decays ~0.5 kcal/mol above r ≈ 10 Å (arrow c) and grows again by the same amount at r ≈ 8 Å (arrow b); ΔV2(r) drops sharply (~1.5 kcal/mol) in the region 6 Å < r < 8 Å, followed by a smaller increment of ~0.5 kcal/mol up to r ≈ 4.5 Å (~rss, arrow a), where it continues to decrease steadily as the monomers approach the close-contact distance rcm. These up-and-down changes result in an overall downward slope of ΔV2(r), which yields a total stabilization of ~1.8 kcal/mol with respect to the salt-free solution (cf. Figure 5). The inset of Figure 1B shows the mean forces exerted by the solvent in pure water and 2 M concentration; the forces are positive at all distances, which means that they tend to separate the monomers at all distances regardless of the ionic strength. The forces exerted by the solvent are calculated as in ref 43, i.e., To further analyze the molecular origin of the changes in ΔV2(r), the spatial structure of the salt-free solvent is analyzed. As the distance between the monomers increases, there is a restructuring of water around the solutes, whose changes can be characterized by analyzing the spatial distribution function (sdf) of the solvent.45 For a given conformation of the solute, the sdf’s are calculated as g(r) = ρw−1ρ(r) = ρw−1δN(r)δV−1, where ρ(r) is the number density of water molecules (water oxygen) at position r; δN(r) is the average number of water molecules within an element of volume δV centered at position r, given by δN(r) = τ−1 ∫N(r,t) dt, with N(r,t) = Σ δ (r – ri(t)), where the sum runs over all of the water molecules in the liquid, and δ (x) is 1 if x
The overall features described above for DR are observed in all the other dimers. However, quantitative differences are also evident that are unique to each case. Figures 2B–4B show ΔV2(r) and Fs,M(r) for SR, DS, and SS, while Figures 7–9 show the location of the corresponding sdf peaks (for r < Rs). Close inspection of these figures shows that Fs,2 decreases with respect to Fs,0 in the region rcm < r < rss in all cases; this behavior is also observed in DK (see below), DH, SK, and SH (not discussed here). Note, however, that the destabilization of the SR dimer at 2 M with respect to pure water (see ΔV2(r) in Figure 2B) results mainly from changes in the relative magnitude and direction of the solvent forces at r > rss. Note that the solvent force above r ≈ 7.5 Å is mostly repulsive in 2 M solution but attractive in pure water, which accounts for large part (~1 kcal/mol) of the total destabilization of the dimer (~1.2 kcal/mol). The sharp changes in ΔV2 observed in the DR dimer are less pronounced in this case, although still appear about the same distances. For the DS dimer in pure water, the solvent exerts a repulsive force (Fs,0 > 0) at all distances (inset in Figure 3B); however, increasing the salt concentration causes the solvent forces to become attractive (Fs,2 < 0) when new sdf peaks form (at r ≈ 4.5 and r ≈ 7.5 Å). Only two peaks are observed in the solvent density around DS (cf. Figure 8); i.e., above Rs ≈ 8–9 Å the monomers become independently hydrated. Figure 3B shows that in this case ΔV2(r) increases steadily only in the region r < rss ≈ 4.5 Å, which is sufficient to account for large part of the overall stabilization of the dimer. Similar behavior is observed for SS (cf. Figure 4B), where ΔV2(r) undergoes significant changes (>RT) only at distances shorter than rss. However, in this case the tendency to stabilize the dimer begins at the position of the second peak at r ≈ 7.5 Å rather than at the first peak at r ≈ 4.5 Å as in DS; however, the appearance of the first peak produces a significant change in ΔV2 of ~0.8 kcal/mol. As shown in the inset of Figure 4B, Fs,2 is not only less repulsive than Fs,0 in the region rcm < r < rss but is also more attractive for rss < r < 7.5 Å; the changes of the relative strengths/directions of the forces in both intervals account for most of the stabilization of the dimer. The observations above indicate that besides the electrostatic effects of bulk a subtle balance of forces operate on the solutes, which originates in the structure of the surrounding solvent (see Discussion). Further insight on these effects may be gained by analyzing the DR and DK dimers since the presence of salts elicits the opposite effects on their PMF (cf. Figure 5). Figure 10 shows ΔV2 and Fs,M(r) for DK. A comparison with Figure 1B reveals a similar up-and-down modulation of ΔV2 as a function of r. However, the relative magnitudes of the decays and growths in each interval result in an overall upward slope of ΔV2, leading to ~1 kcal/mol destabilization, in contrast to DR. The magnitude of Fs,M is larger in DK than in DR, although the solvent tends to dissociate the dimers in both cases (Fs,M > 0
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The effects of salts on the intermolecular potentials between polar/charged amino acid side chains have been studied quantitatively through systematic MD simulations. The strength of the interactions was shown to either increase or decrease with the addition of salt up to 2 M concentration. No obvious correlation was observed between the polar/charged nature of the interacting monomers and the extent of (de)stabilization of the dimers. The results show that a critical balance of solvent forces operates on the solutes in the concentration range studied. The ions appear to affect the mean forces exerted on the solutes at specific intermolecular separations. The changes observed in the solvent forces occur when regions of high solvent density develop between the solutes as they dissociate. The solvent forces, Fs,M, shown in the insets of Figures 1B–4B can be partitioned into two terms: a bulk electrostatic contribution, Fb,M, and a nonbulk (solvation-shell) contribution, Fss,M. Figure 1C shows that both components contribute significantly to the intermolecular potential, both in pure water and in solution. However, the relative magnitudes of the bulk and nonbulk contributions shift within the 0–2 M concentration range (at least in the DR dimer); while in pure water bulk electrostatics are more important than solvent-induced effects, the trend reverses at 2 M concentration. The bulk forces can be further partitioned into contributions from the ions, Fb,i,M, and from water, Fb,w,M. Similarly, the nonbulk forces can be separated into contribution from direct ion–solute interactions, Fss,i,M, and from the hydration water, Fss,w,M. Bulk forces (Fb,M) operate regardless of the salt concentration and provide for a basal modulation of the interactions (e.g., the Debye–Hückel theory46 describes this effect in strong electrolytes). However, the results presented here show that nonbulk contributions are critical to define the outcome of the stabilization, particularly in the upper limits of salt concentrations (~1–2 M). It would be of interest to study the behavior of each of the above contributions with the addition of salt. Such analysis would allow identification and quantitative characterization of the most important forces underlying molecular association/dissociation of small molecules in solution. A small ligand on a protein/solvent interface experiences the same kind of forces (and torques) discussed here; such interactions control the manner in which the ligand docks to the protein and determine the strength of the association. Prediction of ligand-binding modes and calculations of binding free energy are two basic goals in medicinal chemistry for the design or improvement of drugs. Insight into the molecular origins of solvent-induced interactions and their modulations by salts would have an obvious impact in the reliability of such calculations and predictions. Finally, the prospect of describing the complex nonbulk effects of salts, i.e., quantifying Fss,w,M and Fss,i,M, through a simplified picture that may only require knowledge of the location of sdf peaks (of water and ions) around the solutes may have other practical implications. Biological systems vary greatly in size and involve processes that may span several orders of magnitude. Realistic simulations of these systems using an atomistic description of all their constituents (solutes, salts, cosolvents, water, etc.) are computationally expensive. Besides, convergence and statistical significance of thermodynamic quantities extracted from such simulations may also require long simulations (possibly unrelated to the underlying biological process, e.g., calculation of binding free energy or prediction of ligand-binding modes). Replacing the atomistic description of part of the system by a continuum47–49 would allow expansion of the scope of applicability of computer simulations to biological and chemical systems. Describing solvent-induced forces through a simple model that only accounts for the structure (and possibly dynamics) of water and ions in high-density regions around the solutes (e.g., using integral equation-based formalisms50–53) may help to incorporate into such continuum formulation an important component of the physics of the system that is required for meaningful calculations. Corrections for these effects in continuum approximations have largely been ignored (see discussion in refs 47 and 48), and efforts have been directed mainly to describing bulk electrostatic effects. A description of nonbulk effects is ultimately needed for incrementally improving the quality of continuum approximations. | |||||||||||||||||
This study utilized the high-performance computational capabilities of the Beowulf PC/Linux cluster at the National Institutes of Health (NIH) (http://biowulf.nih.gov). The simulations were performed in the Beowulf GNU/Linux cluster at the Center for Molecular Modeling (http://cmm.cit-.nih.gov). This research was supported by the Intramural Research Program of the NIH, through the Center for Information Technology, U.S. Department of Health and Human Services, Bethesda, Maryland. | |||||||||||||||||
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