Trimethylamine‑N‑oxide’s Effect on Polypeptide Solvation at HighPressure: A Molecular Dynamics Simulation StudyRahul Sarma and Sandip Paul*
Department of Chemistry, Indian Institute of Technology, Guwahati Assam, India-781039
ABSTRACT: The solvation characteristics of a 15-residue polypeptide andalso the structure of the solution in the presence and absence oftrimethylamine-N-oxide (TMAO), one of the strongest known proteinstabilizers among the natural osmolytes both at low and high pressures, areinvestigated under high pressure conditions by employing the moleculardynamics simulation technique. The goal is to provide a molecular levelunderstanding of how TMAO protects proteins at elevated pressures. Twodifferent conformations of the polypeptide are used: helix and extended.Analysis of peptide hydration characteristics reveals that the pressure-induced enhancement of hydration number is higher for theextended state as compared to the helix. TMAO shows an opposite effect and causes more dehydration of the extended state.The total number of atomic sites that solvate peptide residues increases in the presence of TMAO, whereas the number ofhydrogen bonds formed by peptide with solution species reduces due to the inability of TMAO to donate its hydrogen to peptidehydrogen bonding sites. In solution, both hydrophobic and hydrogen bonding sites of TMAO are found to be well solvated bywater molecules and solvation of TMAO enhances water structure and reduces the number of nearest identical neighbors forwater. Pressure and TMAO are seen to have counteracting effects on water structural properties. Implications of these results forcounteracting mechanism of TMAO are discussed.
I. INTRODUCTIONProteins are known to show structural changes at high pressure,though the physical basis for the pressure-induced unfolding ofproteins is not clear yet. Numerous studies indicate that, incontrast to the heat-denatured proteins, pressure-denaturedproteins are relatively compact.1 Negative volume change uponpressure unfolding has been observed and likely arises fromcombination of some compensating factors that includeelimination of internal cavities and voids upon disruption offolded structures and exposure of hydrophobic and polarmoieties.2−4 In a NMR study, the hydrogen exchange rate ofproteins is found to be higher under high pressure conditions,suggesting protein denaturation through the penetration ofwater molecules into the tightly packed hydrophobic core ofproteins.5 Molecular dynamics (MD) simulation also showsmore penetrating water molecules for the high pressurestructure of ubiquitin than the low pressure structure, providingevidence for the water penetration model.6 Generally, waterpenetration into the protein interior at high pressure isconsidered as a primary driving force for pressure-inducedprotein structural transition.2,6−10 It is argued that waterpenetration leads to relaxation in water translational restrictioncaused by high pressure, giving an entropic profit to the system,and hence favors the pressure-denatured structure.11
Cosolvents have been shown to affect the unfolding ofproteins by high pressure. While urea, the most commonprotein denaturant, decreases the denaturation pressure,12 thecounteracting effect of trimethylamine-N-oxide (TMAO), oneof the strongest known protein stabilizers among the naturalosmolytes,13 against high pressure has been extensivelyconfirmed in a variety of protein systems.14−16 The question
that arises naturally and also made many researchers interestedover the past few years is, how does TMAO cause proteinstabilization and protect proteins at high pressure? A largenumber of studies have been devoted in this direction, but themechanism has remained somewhat elusive. Multiple proposalshave been put forward that include protein stabilization bypreferential exclusion of TMAO17−21 through alteration ofwater structure22−24 to preferential solvation of TMAO bywater.25 TMAO’s depletion from the protein surface is a well-captured phenomenon,18−21,26,27 but simulation studies alsoindicate a small enhancement in the concentration of TMAOmolecules in the vicinity of hydrophobic solutes28,29 as well asof hydrophobic polymer30 and decaalanine peptide31 in binaryTMAO solution. Therefore, better accommodation of TMAOnear the hydrophobic surface of protein is not unlikely.Interaction of TMAO with positively charged side-chains aswell as with the peptide backbone through its hydrated formhas also been suggested in the literature.32−35 In solution,TMAO is found to interact strongly with water to form di- and/or trihydrated TMAO complexes,24,25,36,37 enhancing the waterstructure at the same time.23−25,32,37−40 Although a physicalmodel for TMAO-induced protein stabilization can be positedin which availability of water to solvate protein residues reducesdue to the solvation of TMAO molecules and, corresponding tothis model, a decrease in the number of protein−waterhydrogen bonds31,32 and dehydration of hydrophobic sol-utes41,42 and carbon nanotubes23 were observed in TMAO
Received: May 27, 2013Revised: June 20, 2013Published: June 26, 2013
solution, by no means is the model sufficient to explain themechanism of action of TMAO. Moreover, different measuresof water structure have been found to display opposite trends inthe presence of TMAO, highlighting the limitations ofarguments that relate enhanced or decreased water structureto macromolecular thermodynamics.24,30,31
As discussed above, a definitive mechanism that can accountfor TMAO’s ability to stabilize proteins and also to counteractthe deleterious effect of pressure is not established yet andexploring the molecular mechanism of TMAO-inducedstabilization and counteraction is the goal of the currentstudy. MD simulations are carried out for a 15-residue modelpeptide in the presence and absence of TMAO at low as well ashigh pressures. Note that pressure-denatured proteins arereported to be relatively compact as compared to heat-denatured proteins, and unlike in the case of high temperature,the molecule cannot be described as a fully extended randomcoil.1 However, to investigate the solvation characteristics offolded and unfolded proteins under high pressure conditionsand the role of TMAO on this protein solvation, we have usedhere two different conformations of the model peptide: helix(denoted as F) and extended (denoted as U). During thesimulation, the peptide is allowed to fluctuate (i.e., flexiblemodel) and the F and U states are ensembles of conformationswhich are “folded” and “unfolded”, respectively. Also importantto note is that only 9 residues (4−12) of the helix are in α-helical regions. Therefore, we focus on solvation of residues 4−12 only, and in this Article, the results reported for peptidesolvation exclude the remaining 6 residues. The followingimportant issues are addressed. First, what is the role of TMAOon the pressure-induced modification of peptide hydration?Can TMAO offset enhanced hydration of the peptide underpressure conditions? Second, how does TMAO interact withpeptide residues and water molecules at high pressure and whatis the implication of TMAO solvation by water in pressure-induced modification of water structural properties?The remainder of this Article is organized into three sections.
The models and simulation details are briefly described insection II, the results are discussed in section III, and ourconclusions are summarized in section IV.
II. MODELS AND SIMULATION METHODClassical MD simulations of a 15-residue model peptide (withtwo different conformations) were carried out in pure water aswell as in binary TMAO solution (≈4 M in strength) at threedifferent hydrostatic pressures: 1, 4000, and 8000 atm. In thecase of pure water (abbreviated as PW), the peptide wassolvated with 1500 water molecules, whereas the aqueousTMAO solution (abbreviated as PT) was generated byreplacing 400 water molecules with 100 TMAO molecules.We used the popular SPC/E model43 for water in all thesimulations, and TMAO was described with the modelproposed by Kast et al.44 Note that the Kast model forTMAO has been used extensively in previous simulations, andalthough a different force field, the “osmotic model”, that showsbetter TMAO exclusion from the vicinity of the protein surfacethan the Kast model, was proposed recently by Garcia and co-workers, no difference was observed using either the Kast or theosmotic model between the folded and unfolded ensembles ofthe protein Trp-cage with respect to their preferentialinteraction with TMAO.45 We have abbreviated atomic sitesin these molecules as Im, where I is the conventional atomicsymbol of that particular atomic site and the subscript m can be
w (for water) and t (for TMAO). For the helical structure ofpeptide, the initial coordinates were taken from the X-raystructure of ribonuclease S (PDB code: 2RNS).46 Afterextracting the first 15 residues, we made a few minormodifications (replacement of residues LYS1, GLU9, andGLN11 by ALA1, LEU9, and GLU11, followed by protonationto give a zwitterionic form with N-terminal NH3
+ and C-terminal COO− groups) to obtain a good starting structure forour simulations. The initial structure of this helix is shown inFigure 1 with the sequence of amino acids. The extended
structure of the peptide (not shown) was generated using thexleap program of AMBER 10.47 Histidine was deprotonated,and the peptide was neutralized by using a Na+ ion. The all-atom parameter set AMBER ff99SB47 was used to describe thismodel peptide.The solution properties were investigated by carrying out
MD simulations at 298 K. All MD runs were performed usingthe AMBER 10 suite of programs.47 To obtain a reasonableinitial structure, we first performed energy minimization (2500steps of steepest descent method followed by 2500 steps ofconjugate gradient method) of the system which was generatedinitially using the packmol program.48 We note that the energyminimization (which relieves bad van der Waals contacts) wasperformed in two steps, first holding the peptide fixed by usingharmonic restraints (force constant = 500.0 kcal mol−1 Å−2)and then removing the restraints on the peptide. After that,each system was heated slowly from 0 to 298 K (to avoid voidformation) for 50 ps in the canonical (NVT) ensemble withweak restraints (force constant = 10.0 kcal mol−1 Å−2) on thepeptide and then equilibrated for 1 ns at the desired pressure inthe isothermal−isobaric (NPT) ensemble without anyrestriction on the peptide. The Berendsen barostat49 wasused to maintain the physical pressure with a pressurerelaxation time of 2 ps. Temperature was controlled by theLangevin dynamics method with a collision frequency of 1 ps−1,and a time step of 2 fs was used in all simulations. Bondsinvolving hydrogen were constrained by applying the SHAKEalgorithm. To remove edge effects, we used periodic boundary
Figure 1. Initial structure of the helical peptide. Sequence: ALA-GLU-THR-ALA-ALA-ALA-LYS-PHE-LEU-ARG-GLU-HIS-MET-ASP-SER. Blue, red, and purple colors are for positive, negative, andnonpolar residues, respectively. The primary structure of the peptide isalso shown.
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conditions. A cutoff radius of 12 Å was applied for non-bondinginteractions, and the long-range electrostatic interactions weretreated using the particle mesh Ewald method. Afterequilibration of 1 ns, simulations were continued for a further10 ns, and the results reported in this Article are from these last10 ns of simulation periods.
III. RESULTS AND DISCUSSIONA. Hydration of Peptide Residues. To obtain molecular
details of peptide hydration at different pressures in thepresence and absence of TMAO, we calculated site−site radialdistribution functions (rdf’s) between peptide and water. Thewater distribution around the peptide heavy atoms is shown inFigure 2. The small first peak at about 2.8 Å is an indicator of
the hydrogen bonding interaction between water and peptideresidues. The second peak at about 3.8 Å, which is related tonon-hydrogen-bonding interaction (e.g., hydrophobic hydra-tion), is much more pronounced than the first peak. The waterdensity in the close proximity of the peptide is found to benoticeably higher for the U state than for the F state. However,the water density near the peptide is lower than that in the bulkregardless of whether the peptide is in the F or U state,indicating exclusion of water from the peptide surface. Theobserved water exclusion is consistent with the notion thatproteins fold spontaneously in nature as water itself is a slightlybad solvent.Considering the effect of pressure alone, i.e., in the absence
of TMAO, we find that the first peak height is unaffected byhigh pressure, whereas the first minimum shows a slight upwardmovement (Figure 2a). On the other hand, increasing thepressure from 1 to 8000 atm leads to a significant modification
of the second peak. To be specific, the peak position shifts to ashorter distance and the peak magnitude increases monotoni-cally with pressure. These findings reveal a negligible effect ofhigh pressure on the tendency of a protein to form hydrogenbonds with water and a compressed water shell in the vicinity ofnon-hydrogen-bonding sites, in accord with the suggestion thatthe water shell is much more compressed in the vicinity ofnonpolar groups as compared to that of hydrogen bondingsites.50,51
Comparison of water pair function around the peptideresidues in water and binary TMAO solution does not revealany counteracting effect of TMAO against pressure. TMAOrather enhances water density near the peptide at low as well ashigh pressures (Figure 2b,c). Water density enhancement byTMAO near the peptide, however, does not necessarily implymore water molecules in the close proximity of the peptideupon addition of TMAO. It only suggests, if anything, that thenumber ratio of vicinal water to bulk water is higher in binaryTMAO solution as compared to pure water. Despite the higherwater density in the nearby region of peptide, dehydration ofpeptide occurs (as shown later) in the presence of TMAO.The hydration characteristics of the peptide residues were
probed further by calculating the number of water moleculesaround the peptide residues as well as the number of peptide−water hydrogen bonds in different environments. The numberof water molecules in the first solvation shell (FSS) wascalculated first for each of the 4−12 residues, and then thosenumbers were added to obtain the total hydration number. Awater molecule was considered to be inside the FSS when itsoxygen atom fell within a distance of 3.5 Å from any heavyatom of the selected peptide residue. On the other hand, ahydrogen bond, D−H···A, was defined by a maximal distance of3.5 Å for D−A (where D is donor and A is acceptor) and amaximal angle of 45° for H−D−A.The average number of water molecules in the FSS of the
peptide is assembled in Table 1. As expected from the peptide−
water rdf, we find more water molecules in the FSS of peptideresidues in the U state. The hydration number increases withpressure for both the F and U states. Interestingly, pressure-induced enhancement of peptide hydration is higher for the Ustate, which is clearly demonstrated in Figure 3. For our modelpeptide, the hydration number in pure water is enhanced byabout 22 and 31, respectively, for the F and U states as thepressure increases from 1 to 8000 atm (Table 1). On thesegrounds, we can infer that high pressure stabilizes both the Fand U states through enhancement of short-range interactionswith water molecules and the relative stabilization is higher forthe later conformation. It is remarkable here that extensive MD
Figure 2. Distribution of water oxygen around the peptide heavyatoms (residues 4−12) as a function of pressure in pure water (a) andalso as a function of TMAO concentration at 1 (b) and 8000 (c) atmpressures for the F (left) and U (right) states. Black, red, and blue linesare for 1, 4000, and 8000 atm in pure water. For the TMAO system,cyan and orange colored lines are used.
Table 1. Average Number of Water and TMAO HeavyAtoms within 3.5 Å of Any Heavy Atom of PeptideResiduesa
aNW, NN, NC, and NO represent the number of water oxygen andnitrogen, carbon, and oxygen of TMAO, respectively.
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simulations on proteins suggested pressure denaturation due tothe higher compressibility of the hydration shells of unfoldedproteins than the folded ones.51 Table 1 shows TMAO’s abilityto remove water molecules from the close proximity of thepeptide. While dehydration in the presence of TMAO occursregardless of whether the peptide is folded or unfolded, for aparticular pressure, the effect is relatively higher for the U state(Table 1). Thus, TMAO can offset the pressure-inducedrelatively higher stabilization of the extended state as comparedto the compact state by causing more reduction of short-rangeinteractions with water molecules for the former.In Table 2, we have presented the number of peptide−water
hydrogen bonds together with the relative population of wateroxygen in the FSS of peptide residues that are engaged inhydrogen bonding interactions with the peptide. The peptide−water hydrogen bond number is higher for the U state, asexpected from enhanced hydration. Also consistent with the
effect of pressure on hydration of peptide residues is theobservation that, in pure water, the hydrogen bond numberincreases with pressure. The enhancement in hydrogen bondnumber, however, does not differ much for the F and U states,as shown in Figure 4. It is also important to emphasize that the
relative population of hydrogen bonded water molecules in theFSS decreases with pressure for both conformations of thepeptide (Table 2). Thus, the U state does not gain much netstabilization over the F state at high pressure from peptide−water hydrogen bonding interactions. In other words, intra-protein hydrogen bonds are likely to be preserved even atelevated pressure. This is consistent with the observation thatpressure-denatured structures are much more compact than thethermally denatured structures.1 On the other hand, hydrationof peptide residues is higher for the extended state (asdiscussed above). This leads us to suggest that proteindenaturation by high pressure is caused by the enhancedhydration of non-hydrogen-bonding sites. Note that, in a seriesof studies, we showed efficient packing of water moleculesaround hydrophobic solute at high pressures and consequentpressure-induced relative stabilization of the water-separatedconfiguration of nonpolar solute as compared to its associatedstate.42,53 Also, it has been reported in the literature that, whileexposure of hydrophobic groups to water is highly favoredunder pressure, high pressure has little effect on the tendency ofa protein to form hydrogen bonds.52
Variation in peptide−water hydrogen bond number withpressure is insignificant in the presence of TMAO, though thereis clear reduction in the relative population of hydrogen bondedwater molecules in the FSS at high pressure for both the F and
Figure 3. Top: Number of solution species (water and TMAO) in theFSS of peptide residues (left) and total number of contacts betweenpeptide and solution species (right) for the F (●) and U (■) states inpure water (black) as well as in binary TMAO (red) solution. Bottom:Difference between U and F states.
Table 2. Number of Hydrogen Bonds Formed by PeptideResidues (4−12) with Water (nHB
aThe numbers in parentheses give relative populations (in percentage)of water and TMAO oxygen in the FSS of peptide residues that areengaged in hydrogen bonding interactions with peptide.
Figure 4. Top: Total number of hydrogen bonds (NHB) formed bypeptide residues with solution species (left) and the relativepopulation of heavy atoms in the FSS of peptide residues that areengaged in hydrogen bonding interaction with peptide (right) for theF (●) and U (■) states in pure water (black) as well as in binaryTMAO (red) solution. Bottom: Difference between U and F states.
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U states (Table 2). TMAO shows a tendency to reduce theefficiency of the peptide atomic sites to form hydrogen bondswith water molecules, as we predicted in a recent study.54 Theeffect is higher for the U state and increases with pressure.Thus, through its effect on peptide hydration, TMAO can causerelatively higher destabilization of the U state both at low andhigh pressures. Though this is consistent with TMAO-inducedprotein stabilization, for a more clear picture, one needs toconsider interaction between protein and TMAO as well andwhat follows is the discussion of this interaction.B. TMAO’s Interaction with Peptide Residues. Solvation
of peptide residues by TMAO was probed by calculating thenumber of TMAO atomic sites around the peptide residues. Asin the case of water, the number of TMAO atomic sites in theFSS was calculated for each residue using VMD55 byconsidering a heavy atom to be inside the FSS when it fallswithin a distance of 3.5 Å from any heavy atom of the selectedpeptide residue. Table 1 presents the total number of TMAOatomic sites in the FSS of the peptide. We first concentrate onthe nitrogen atom of TMAO, which, being the central atom ofthis osmolyte, is generally considered to describe theinteraction of protein residues with TMAO molecules. Thenumber of TMAO nitrogen molecules in the FSS of eachresidue is very small and, overall, there is only about 0.1nitrogen in the close proximity of the peptide at 1 atm (Table1). While this number is much smaller than the number ofwater molecules in the FSS and is related to the lower numberdensity of TMAO in the system (the probability of finding amolecule near a target molecule increases with concentration),it is inappropriate to say that the large reduction is only due tothe lower number density of TMAO in the system. We findthat the proportion of water to TMAO in the FSS is muchhigher than the bulk proportion, implying preferential exclusionof TMAO in that region. Also needed to mention here is thepopular notion that TMAO is excluded preferentially from theprotein surface.17−21 Thus, the negligible number of TMAOnitrogen in the FSS of the peptide residue relative to water isalso contributed by some other factors that definitely includehigher excluded volume for a TMAO molecule (the vdW radiifor water and TMAO are about 1.556 and 2.95,56 respectively).This is further reflected in the reduced number of solutionspecies, NSOL (=NW + NN, where NW and NN are the averagenumber of water oxygen and TMAO nitrogen, respectively), inthe FSS in binary TMAO solution than in pure water (Figure3). Because of a larger surface area, NSOL is higher in the U stateas compared to that in the F state, but interestingly, thedifference in NSOL between the F and U states, i.e., ΔNSOL =NSOL
U − NSOLF , is lower in binary TMAO solution than in pure
water. This is in qualitative agreement with previous estimationof excluded volume change upon unfolding.56 In particular,Schellman noted that excluded volume, which is invariablypositive for unfolding (increased surface area) in all cosolvent−protein interactions and stabilizes the folded form, is the majorcontributing factor for TMAO-induced protein stabilization.56
Note, however, that, although higher excluded volume ofTMAO does not allow its central atom (Nt) to penetrate in aspecific region around the protein surface, causing relativedestabilization of the unfolded state, TMAO interaction withprotein residues is possible through methyl groups and oxygenatom, and these interactions can contribute to relativestabilization of the unfolded state. For our model peptide,TMAO interaction occurs mainly through methyl groups(Table 1). The number of oxygen atoms in the FSS of peptide
residues is also significantly higher than that of nitrogen atoms.The presence of all these sites increases the total number ofheavy atoms, NCON (=NW + NN + NC + NO, where N is theaverage number with subscripts W, N, O, and C standing foroxygen of water and nitrogen, oxygen, and carbon of TMAO,respectively), in the FSS in binary TMAO solution than in purewater (Figure 3). Nonetheless, the relative enhancement inNCON is lower in the U state as compared to that in the F state(compare magnitudes of ΔNCON = NCON
U − NCONF in Figure 3),
which is an indication of depletion of interacting sites next tothe extended form relative to the folded form. Therefore, whilethe addition of TMAO to pure water seems to stabilize boththe F and U states of the peptide through enhancement ofshort-range interactions between peptide and solution species,the relative stabilization is higher for the helix as compared tothe extended form. This looks surprising at first instance butcorresponds well with the data obtained by Kokubo et al.31
performing solvation free energy calculations. Their replica-exchange λ sampling simulations clearly showed TMAO-induced enhancement of the favorable vdW solvation freeenergy of decaalanine peptide for helix and denaturedconformations, with the helix being the more stable form inbinary TMAO solution vs water.31
In a recent article, using N-methylacetamide as a model ofthe peptide backbone, we showed that TMAO can interact withbackbone oxygen through its methyl groups.54 Hydrogenbonding interactions between these atomic sites are, however,unlikely. We predicted that, due to the presence of TMAOmethyl groups near these hydrogen bonding sites, theirefficiency for hydrogen bond formation decreases. On theother hand, TMAO oxygen is expected to form some hydrogenbonds with backbone nitrogen and positive side-chains ofpeptide residues. We, therefore, calculated hydrogen bondnumbers between TMAO oxygen and peptide residues usingthe geometric criteria defined for peptide−water hydrogenbond calculations. Table 2 presents the numbers obtained forthe peptide residues. We can see only a small number ofhydrogen bonds between peptide and TMAO. The probabilityof TMAO oxygen in the FSS to engage in hydrogen bondinginteraction with peptide residues increases for the U state, andthe probability decreases with pressure (see relative populationin Table 2). Because TMAO does not donate its hydrogen topeptide hydrogen bond acceptor sites, the total number ofhydrogen bonds (NHB) formed by peptide residues withsolution species decreases mostly in the presence of TMAO(Figure 4). Figure 4 shows a TMAO-induced noticeabledecrease in relative population of heavy atoms that make directcontact with peptide residues through hydrogen bondinginteractions. Additionally, TMAO-induced reduction in NHBis higher in the U state as compared to that in the F state(compare the magnitudes of ΔNHB = NHB
U − NHBF ), implying
relatively higher destabilization of the extended state. Thus, theenhancement of short-range vdW interactions in TMAOsolution is likely to stabilize the peptide, whereas the peptideis destabilized by reduction of hydrogen bonding interactions.The most significant observation is that both of these factorsfavor the folded state over the extended state.
C. Solvation of TMAO. Behavior of water molecules in thevicinity of TMAO atomic sites was examined to obtaininformation regarding the hydration of TMAO under highpressure conditions. The distribution of water around TMAOoxygen and carbon atoms is displayed in Figure 5, whereas thenumber of water molecules (central atom only) that are within
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3.5 Å of these TMAO atomic sites is tabulated in Table 3. Thetypical surface-parallel orientation of water molecules in the
vicinity of a hydrophobic group28,29 can be seen in the Ct−Owand Ct−Hw rdf profiles. There arises a tail of hydrogen densityat shorter separation, and the region of maximum density issimilar for both oxygen and hydrogen of water. We, however,note that, relative to that in the Ct−Ow rdf, the first peak in Ct−Hw rdf is located at a slightly longer distance, which is differentfrom that observed in the case of nonpolar solute28,29 but isconsistent with the results reported by Athawale et al.30
High pressure modifies the water distribution in the vicinityof TMAO methyl groups significantly. In particular, the firstmaximum and minimum shift to shorter distances at highpressure, indicating inward movement of the first hydrationshell. The monotonic increase of the first peak and decrease ofthe first minimum with pressure imply a movement of watermolecules from the large-r side of the first hydration shell
toward its short-r side. The water movement under pressurethus leads to a reduction in empty spaces in the close proximityof TMAO methyl groups with higher water density in thisregion and also makes the hydration shell more structured.Pressure-induced enhanced packing efficiency of watermolecules around hydrophobic solute has been reported inthe literature.7,53,57,58 As expected from the monotonic increasein the height of the first peak with pressure with compressedwater shell, the average number of water molecules in the closeproximity of a methyl group increases as the pressure isincreased. Table 3 reveals that the number of water moleculeswithin 3.5 Å of a single methyl group increases by about 55% asthe pressure increases from 1 to 8000 atm.The hydrogen bonding interaction between TMAO oxygen
and water is demonstrated in the Ot−Ow and Ot−Hw rdfprofiles. In particular, the contact peaks in Ot−Ow and Ot−Hwrdf profiles appear at about 1.8 and 2.8 Å, respectively, asreported in a previous MD simulation study.30 Considering theeffect of pressure, we find that the first peak in the Ot−Ow rdf isnot affected much at high pressure, but the first minimumshows an inward and upward movement, revealing a tendencyof part of the hydration water to move toward the large-r sideof the first shell under high pressure conditions. The behaviorof water in the vicinity of TMAO oxygen at high pressure isthus quite different from that in the vicinity of TMAO methylgroups where water molecules move from the large-r side of thefirst shell toward its short-r side. In other words, the water shellis much more compressed in the close proximity of nonpolargroups relative to the water shells surrounding hydrogenbonding sites. As shown in Table 3, the number of watermolecules within a distance of 3.5 Å of the TMAO oxygenincreases from 2.7 at 1 atm to 3.3 at 8000 atm (22% increase).This relative enhancement is much smaller than that aroundmethyl groups. The significant pressure-induced modificationof the hydrophobic hydration shell is also reflected in Table 3,which shows a lesser number of water molecules near eachmethyl group (relative to TMAO oxygen) at 1 atm but morewater molecules for the same at 8000 atm.The number of hydrogen bonds between TMAO oxygen and
water was also computed by imposing cutoff distances of 2.5and 3.5 Å, respectively, for Ot−Hw and Ot−Ow and asimultaneous cutoff angle of 45° for Hw−Ow−Ot. Figure 6illustrates the fraction of molecules ( f n) that engage in n
Figure 5. TMAO oxygen−water oxygen (top) and TMAO carbon−water oxygen (bottom) site−site rdf’s as a function of pressure for theF (left) and U (right) states. Different colors are as in Figure 2.Dashed lines are for TMAO oxygen−water hydrogen (top) andTMAO carbon−water hydrogen (bottom) rdf at 1 atm.
Table 3. Average Number of Water Molecules within 3.5 Åof TMAO Carbon (NCt) and Oxygen (NOt) Together withthe Number of Hydrogen Bonds between TMAO Oxygenand Water (nHB) and Their Ratios
Figure 6. Fraction of TMAO molecules that engage in n number ofhydrogen bonds with water molecules for the F (left) and U (right)states. Different colors are as in Figure 2.
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number of hydrogen bonds, and the average number ofhydrogen bonds per TMAO molecule is depicted in Table 3.The majority of TMAO oxygens are found to have either 2 or 3hydrogen bonds to water (Figure 6), and on average, there areabout 2.1 water molecules which are hydrogen bonded to eachTMAO oxygen at 1 atm (Table 3). The existence of 2.1hydrogen bonds between TMAO oxygen and water, whichcorresponds well with previously reported two to threehydrogen bonds between water and TMAO,31,35,37,59,60 impliesabout 76% water molecules within 3.5 Å of TMAO oxygen thatdonate hydrogen bonds. Note that the percentage of hydrogenbonded water is higher than that in the case of the watersolvation shell (see below). High pressure decreases the relativepopulation of the 2-coordinated water molecule and increasesthose of 3- and 4-coordinated water molecules for TMAOoxygen. Although this is consistent with pressure-inducedenhanced hydration of TMAO oxygen, the relative enhance-ment in hydrogen bond number is not very significant (onlyabout 3%). Additionally, the relative population of watermolecules in the close proximity of TMAO oxygen thatparticipate in hydrogen bonding interaction with TMAOoxygen decreases with increasing pressure (Table 3). Theexamination of solvation characteristics of TMAO thus suggeststhat both hydrophobic and hydrogen bonding sites of TMAOare well solvated by water molecules. The hydration numberincreases with increasing pressure for both of these atomic sites,though the enhancement is much lower for TMAO oxygen andTMAO−water hydrogen bond number is almost unaffected bypressure.D. Water Structural Properties. The indirect effect of
TMAO solvation on water structural properties was examinedto shed light on the counteracting mechanism. For thispurpose, we calculated the water oxygen−oxygen (Ow−Ow) rdfas well as the water−water hydrogen bond number at differentpressures in the presence and absence of TMAO. In pure waterat 1 atm, the first and second peaks in the Ow−Ow rdf (Figure7) appear at about 2.8 and 4.5 Å, which are characteristic peaksfor the hydrogen bonded first neighbor and the tetrahedrallylocated second neighbor of water molecules. Integration of therdf to 3.5 Å yields 5.1 identical neighbors for each watermolecule (Table 4). For comparison, a previous simulationstudy indicated the existence of 5.2 water molecules (TIP3Pmodel) in the first coordination shell of a water molecule at 1atm and 300 K.61 Among the 5.1 first shell water molecules inpure water at 1 atm, about 3.3 are hydrogen bonded to thecentral water molecule. Thus, a large number (66% to be exact)of first shell water molecules are engaged in hydrogen bondinginteraction with the central water molecule. Consistent withpreviously reported pressure-induced modification of TIP3Pwater structure,58 we observe that, while the location and theheight of the first peak remain practically unchanged, thesecond peak disappears at high pressure (Figure 7). Thus, athigh pressure, part of the water molecules in the second shellmove toward the first shell, filling the empty spaces in thisregion. On one hand, this movement increases crowding ofwater molecules under pressure, and on the other hand, it leadsto a collapse of the water second shell. We find that an increaseof pressure from 1 to 8000 atm enhances the number of wateridentical neighbors by about 35% (Table 4). Water crowding athigh pressure has been reported extensively in the liter-ature.61−63 Table 4 shows pressure-induced enhancement inwater−water hydrogen bond number, which is expected on theface of increased water crowding. Interestingly, the relative
population of first shell water molecules that do not participatein hydrogen bonding interaction with the central watermolecule increases with increasing pressure. It is not hard toassume that one can remove the first shell water molecules thatare not associated through hydrogen bonds relatively easilyfrom the water solvation shell. Since availability of such “free”water molecules increases at high pressure, hydration of proteinresidues should increase with increasing pressure as it allowswater molecules to move freely in the bulk, giving an entropicprofit to the system without disturbing the water hydrogenbonding network much (less energy loss). This is exactly whatwe have observed for our model peptide (as discussed above).Also relevant here is the notion that relaxation in translationalentropy in bulk water arising from water penetration into theprotein interior contributes significantly to protein denaturationat high pressure.11,64,65
Figure 7. Water oxygen−oxygen site−site distribution functions as afunction of pressure in pure water (a) and also as a function of TMAOconcentration at 1 (b) and 8000 (c) atm pressures for the F (left) andU (right) states. Different colors are as in Figure 2.
Table 4. Average Number of Water Molecules within 3.5 Åof Water Oxygen (NH) Together with the Number ofHydrogen Bonds between Water Molecules (nHB) and TheirRatios
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Compared to that for pure water at a particular pressure,TMAO increases the first peak and makes the first and secondwater shells more defined. Enhancement of the first peak heightin the presence of TMAO was also observed in earliersimulations both for TIP3P23 and SPC/E water30 and is relatedto TMAO’s ability of making the water hydrogen bondingnetwork tightly coordinated. Experimental and MD simulationstudies suggested that TMAO can serve as a “water structuremaker” at 1 atm,22−24,32,37−40,66 and our simulation results heresuggest validity of this water structure making property ofTMAO under high pressure conditions as well (see the bottompanel in Figure 7). The number of identical molecules in thesolvation shell of water, however, decreases upon addition ofTMAO (Table 4), which is due to reduced water numberdensity in the system. For our systems, TMAO-inducedreduction in water number is found to be about 20%. As aconsequence of the loss of water molecule in the solvation shell,the water−water hydrogen bond number decreases in presenceof TMAO. Table 4 shows that water loses about 0.5 hydrogenbonds to identical species when TMAO is added to the purewater system. On the other hand, TMAO enhances the relativepopulation of first shell water molecules that are associatedthrough hydrogen bonds, thereby increasing the penalty ofremoving a water molecule from the water solvation shell (largeenergy loss). TMAO’s effect on the water network is, thus,completely opposite to that of high pressure, indicating thatTMAO may offset pressure-induced enhanced hydration ofprotein residues through its counteracting effect on waterstructure. Recently, Schroer et al.16 predicted that it is theindirect effect of TMAO on the water structure that plays amajor role in the protein stabilization by TMAO under highpressure conditions. The results presented here lend support totheir predictions.The fraction of water molecules ( f n) that engage in n number
of hydrogen bonds with identical species is illustrated in Figure8 for a better idea of water hydrogen bonding interactionsunder high pressure conditions in the presence and absence ofTMAO. As expected, water molecules are largely engaged ineither three or four hydrogen bonds and high pressureenhances the fraction of 5-coordinated water molecules,simultaneously reducing the number of lower (2 and 3)coordinated water molecules. What TMAO does on the waterhydrogen bond network is completely opposite to that ofpressure. Due to the large size of TMAO, its solvation in waterfirst requires creation of a large cavity. This results in a dramaticloss in the number of higher (4 and 5) coordinated watermolecules, increasing the fraction of lower (1−3) coordinatedwater molecules. Thus, in agreement with previous MonteCarlo simulation results for the methane−water system atdifferent pressures,67 we observe pressure-induced enhance-ment of higher coordinated water molecules. The additionalevidence here is that TMAO has an opposite effect on the waterhydrogen bonding network, which supports further theimportance of TMAO’s indirect effect on water structure inprotein stabilization by TMAO under high pressure conditions.
IV. SUMMARY AND CONCLUSIONSEmploying the MD simulation technique, the solvationcharacteristics of a polypeptide and also the structure of thesolution in the presence and absence of TMAO wereinvestigated under different pressure conditions to explore themechanism of protein protection by TMAO at low as well aselevated pressures. Computations of site−site rdf’s indicated a
negligible effect of high pressure on the tendency of a proteinto form hydrogen bonds with water and a compressed watershell in the close proximity of non-hydrogen-bonding sites.Pressure-induced enhancement of hydration number was foundto be higher for the U state as compared to the F state. Theseobservations are consistent with the suggested much highercompressibility of the water shell in the vicinity of nonpolargroups as compared to that of hydrogen bonding sites50,51 andalso with the notion that the hydration shells of unfoldedproteins are more compressible than those of folded ones,contributing to pressure denaturation.51 However, for ourmodel peptide, we did not observe much difference in peptide−water hydrogen bond number enhancement between the F andU states, making pressure-induced protein denaturationthrough enhancement of protein−water hydrogen bonds anunlikely possibility. Instead, we observed a negligible effect ofpressure on the tendency of a protein to form hydrogen bonds,as was indicated previously.52 TMAO did not prevent pressure-induced enhancement of water density in the close proximity ofthe peptide residues. However, there was a lesser number ofwater molecules in the peptide hydration shell in aqueoussolution of TMAO and the number of hydrogen bondsbetween peptide and water reduced.While the total number of solution species near the peptide
reduced in binary TMAO solution as compared to pure waterregardless of whether the peptide is folded or unfolded, relativereduction was found to be higher for the U state. Thisobservation corresponds well with the notation that TMAOinduces protein stabilization due to large excluded volumechange upon unfolding.56 We, however, note that TMAO-induced peptide dehydration is not due to the larger excluded
Figure 8. Fraction of water molecules that engage in n number ofhydrogen bonds with identical species as a function of pressure in purewater (a) and also as a function of TMAO concentration at 1 (b) and8000 (c) atm pressures for the F (left) and U (right) states. Differentcolors are as in Figure 2.
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volume of TMAO but is only because of the existence ofTMAO atomic sites (methyl groups in particular) in the closeproximity of the peptide. Our simulations showed TMAOinteracting directly with peptide residues through its methylgroups and oxygen atom, and the total number of heavy atomcontacts between peptide and solution species increased in thepresence of TMAO. Nonetheless, relative enhancement inheavy atom contact was lower for the U state, contributing torelative stabilization of the F state. On the other hand, TMAOwas observed to reduce the efficiency of the peptide to formhydrogen bonds with solution species. The reduction washigher for the U state (relative to the F state), thereby favoringthe folded state further.Investigation of TMAO hydration under high pressure
conditions indicated that the hydration number for bothhydrophobic and hydrogen bonding sites of TMAO, which arewell solvated by water molecules even at 1 atm pressure,increases with pressure, though the enhancement is much lowerfor TMAO oxygen, and the TMAO−water hydrogen bondnumber is almost unaffected by pressure. TMAO and highpressure were seen to have counteracting effects on waterstructural properties. In particular, pressure enhanced numberof nearest neighbor water molecules and, although the water−water hydrogen bond number increased, the relative number offirst shell water molecules that participate in hydrogen bondinginteractions reduced with pressure, indicating pressure-inducedcrowding of water molecules with a relatively destabilized waterhydrogen bond network. On the contrary, TMAO not onlyreduced the number of nearest identical neighbors of water butalso enhanced the relative population of first shell watermolecules that participate in the water hydrogen bond network.From our systematic investigation, we interpret the
mechanism of pressure-induced protein denaturation andTMAO-induced counteraction as follows. Extreme watercrowding at high pressure restricts water movement in thebulk, and for translational relaxation, the relatively “free” watermolecules in the water first shell move to the protein surface.Consequent enhancement of short-range vdW interactionsstabilizes the folded state as well, but the relative stabilization ishigher for the pressure-denatured structure. Hydrogen bondinginteractions between protein and water may not affect thefolded ⇌ unfolded equilibrium under these harsh conditions,and protein denaturation by high pressure is most likely to beassociated with enhanced hydration of non-hydrogen-bondingsites, which results in a relatively compact structure ascompared with the thermally denatured structure. Extendingto the counteracting pathway of TMAO, TMAO can protectproteins at high pressure by preventing water from solvatingprotein residues and also interacting relatively inefficiently withthe unfolded protein. Focusing on the first, TMAO forms somestrong hydrogen bonds with water in the bulk and solvation ofTMAO reduces pressure-induced crowding of water moleculesas well as the number of relatively “free” water molecules. Thisreduces the availability of water molecules that solvate proteinresidues. Another important factor that makes TMAO the mosteffective pressure-counteractant is its inability to interactefficiently with the unfolded state. The total number ofcontacts between protein and solution species increases forboth the F and U states upon addition of TMAO, and hence,both states are likely to be stabilized in aqueous TMAOsolution through short-range vdW interactions. However, therelative stabilization is lower for the unfolded state. Addition-ally, due to its inability to donate hydrogen to protein hydrogen
bonding sites, protein loses some hydrogen bonds to solutionspecies in the presence of TMAO. This hydrogen bondreducing effect, which can easily be considered as a proteindestabilizer, is higher for the unfolded state. TMAO shifts thefolded ⇌ unfolded equilibrium toward the folded state byrelatively higher destabilization of the unfolded state. Hence, wewould like to conclude that the ability of TMAO to offsetprotein denaturation under high pressure conditions arises fromcombination of two effects: (a) TMAO solvation in the bulkwith associated enhancement of water structure, therebypreventing water molecules from solvating protein residues,and (b) inability of TMAO to stabilize the unfolded statethrough its direct interaction with protein residues.
■ ACKNOWLEDGMENTSFinancial support from the Council of Scientific and IndustrialResearch (CSIR) and Department of Science and Technology(DST), Govt. of India, are gratefully acknowledged.
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