Exploring the Molecular Mechanism of Trimethylamine‑N‑oxide’sAbility to Counteract the Protein Denaturing Effects of UreaRahul Sarma and Sandip Paul*
Department of Chemistry, Indian Institute of Technology, Guwahati Assam, India-781039
ABSTRACT: Protein denaturation in highly concentratedurea solution is a well-known phenomenon. The counteractingeffect of a naturally occurring osmolyte, trimethylamine-N-oxide (TMAO), against urea-conferred protein denaturation isalso well-established. However, what is largely unknown is themechanism by which TMAO counteracts this denaturation. Toprovide a molecular level understanding of how TMAOprotects proteins in highly concentrated urea solution, wereport here the structural, energetic, and dynamical propertiesof N-methylacetamide (NMA) solutions that also contain ureaand/or TMAO. The solute NMA is of interest mainly because it contains the peptide linkage in addition to hydrophobic sitesand represents the typical solvent-exposed state of proteins. Molecular dynamics computer simulation technique is employed inthis study. Analysis of solvation characteristics reveals dehydration of NMA and reduction in hydrogen bond number betweenNMA oxygen and water upon addition of TMAO. The effect of TMAO on NMA−urea interaction is found to be insignificant.Because TMAO cannot donate its hydrogen to NMA oxygen, the total number of hydrogen bonds formed by NMA oxygen withsolution species decreases in the presence of TMAO. In solution, TMAO is found to interact strongly with water and urea.Solvation of TMAO makes the water hydrogen bonding network relatively stronger and reduces relaxation of urea−waterhydrogen bonds. Implications of these results for counteracting mechanism of TMAO are discussed.
I. INTRODUCTIONNatural osmolytes, small organic molecules accumulated byorganisms in response to osmotic stress, are known to affect thestability, structure, and function of proteins. In particular, it hasbeen known for many years that a high concentration of urea,which is one of the most commonly available osmolytes, cancause the denaturation of proteins in solution1 and hence caninhibit many important biological processes. Based on manyexperimental and theoretical studies, two mechanisms,“indirect” and “direct” interaction models, have been positedfor urea-conferred protein denaturation. The indirect mecha-nism presumes that urea acts as a structure breaker for water soas to enhance hydration of protein sites.2−7 On the other hand,the direct mechanism hypothesizes that urea moleculespreferentially bind to protein through their stronger inter-actions with protein backbone or side chains than water.8−22
Despite the extensive studies from both experiment and theoryin the past several decades, it is not clear whether the direct orindirect effect provides the driving force in urea-inducedprotein denaturation. In all likelihood, urea denaturation ofproteins results from combination of both direct and indirecteffects.23−27
In contrast to the protein denaturing effect of urea, someother osmolytes are known to bias the unfolded structuretoward the folded state. Trimethylamine-N-oxide (TMAO)represents the extreme among these osmolytes and hasgenerated considerable research interest to the community ofbiophysicists and biochemists over the past few years. Thiscompound is particularly known for its ability to stabilize
proteins28,29 and nucleic acids,30 correct medicinally significantissues, such as prion aggregation31 and cellular folding defects,32
and counteract protein denaturation by urea,1,33 heat, andpressure.34 Although TMAO’s ability to counteract urea-induced protein denaturation at physiological concentrationsof urea to TMAO concentration of 2:1 is well-established,1,33
what is still under debate is the mechanism by which TMAOstabilizes proteins and counteracts urea-conferred proteindenaturation. Multiple proposals have been put forward andthey range from preferential exclusion of TMAO from theprotein35 through alteration of water structure36 to preferentialsolvation of TMAO by water and urea.37 There is generalagreement on the preferential TMAO exclusion from theprotein surface.35,38−42 However, the preferential exclusionmodel does not explain the role of water in solvation of proteinsites. Due to the exclusion of TMAO from the proximity of theprotein surface, inclusion of water in the surface of the proteinbecomes inevitable (termed preferential hydration). If theprotein residues are considered to be better hydrated in TMAOsolution as compared to pure water or if there is little differencein protein hydration shell between pure water and TMAOsolution, then, the question that arises naturally is, why does anunfolded protein shift to its folded state in the presence ofTMAO? Clearly, the preferential exclusion model alone is notsufficient to answer this question.
Received: February 19, 2013Revised: April 13, 2013
The preferential solvation model37 in which water and ureaprefer to solvate TMAO over protein residues seems useful atfirst instance, and corresponding to this model, a decrease inthe number of peptide−water hydrogen bonds43 anddehydration of a carbon nanotube25 were observed in TMAOsolution. However, the model requires removal of urea from theprotein solvation shell which is against the notation that theefficiency of an osmolyte to act on the peptide is not interferedby the presence of a competing osmolyte.44,45
There are also clear discrepancies in simulation studies thatinvestigate the indirect effect of TMAO on water structure.Enhancement of water structure by TMAO in the form ofstronger water−water hydrogen bonds and long-range spatialordering of the solvent was observed in previous simulationstudies.23,25,36 Wei et al. mentioned a significant indirect effectof TMAO on the protein backbone hydration.23 In a latersimulation study, a correlation between water structureenhancement and dehydration of carbon nanotubes in thepresence of TMAO was indicated.25 Some other studies, on theother hand, questioned the usefulness of the water structureperturbation ideas in predicting protein stability.43,46 Inparticular, Athawale et al. found that different measures ofwater structure can display opposite trends in the presence ofTMAO, highlighting the limitations of arguments that relateenhanced or decreased water structure to macromolecularthermodynamics.46
As discussed above, a definitive mechanism that can accountfor TMAO’s ability to stabilize proteins and also to counteractthe deleterious effect of urea is not established yet. So, the goalof the current study is to explore the molecular mechanism ofstabilization and counteraction by TMAO. Transfer free energymeasurements showed that the protein backbone plays thedominating role in determining the extent of proteinstabilization or destabilization by osmolytes and the side-chainsplay only a minor role, and consequently, it was concluded thatthe osmolyte effect operates predominantly on the proteinbackbone.29,47,48 Keeping this in mind and to reduce complexinteractions among solvent, osmolytes, and the various groupsof proteins, MD simulations are carried out for N-methylacetamide (NMA) in pure water as well as in binaryand ternary solutions of urea and TMAO. The solute NMA isthe smallest amide that contains the peptide linkage and,additionally, two hydrophobic sites. To provide a sufficientnumber of NMA solvation sites for water and TMAOmolecules avoiding NMA−NMA hydrogen bonds as much aspossible, dilute NMA solutions are used in this study. Thesystems considered here, therefore, have a negligible number ofNMA−NMA hydrogen bonds and represent typical solvent-exposed states of proteins where protein backbone can freelyinteract with the surrounding solvent molecules. Variouspossible interactions involving NMA, water, and osmolytesare examined first. We then study the solvation of TMAO bywater and urea with an attempt to dissect the counteractingmechanism.The remainder of this article is organized into three parts.
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 METHODTo understand the mechanism of protein protection by theosmolyte TMAO against urea denaturation, we carried outclassical MD simulations of NMA in pure water as well as in
binary and ternary solutions of urea and TMAO. Twenty NMAmolecules (mole fraction of 0.04) were used in each simulatedsystem. An overview of simulations is presented in Table 1.
Note that the total number of molecules was fixed at 500 in allcases and osmolyte solutions were constructed by replacingwater molecules with osmolyte. We used the popular extendedsimple point-charge (SPC/E) model49 for water and describedTMAO and NMA with the rigid version of models proposed byKast et al.50 and Jorgensen and Swenson,51 respectively. Theso-called Duffy-Kowalczyk-Jorgensen (DKJ) model52 wasadopted for urea. The interaction between atomic sites oftwo different molecules was expressed as
εσ σ
= − +αβ αβ αβαβ
αβ
αβ
αβ
α β
αβ
⎡
⎣⎢⎢⎛⎝⎜⎜
⎞⎠⎟⎟
⎛⎝⎜⎜
⎞⎠⎟⎟
⎤
⎦⎥⎥u r
r r
q q
r( ) 4
12 6
(1)
where rαβ is the distance between atomic sites α and β, and qα isthe charge of the site α. The Lennard-Jones (LJ) parameters σαβand εαβ were obtained by using the combining rules σαβ = (σα +σβ)/2 and εαβ = (εαεβ)
1/2. The values of the LJ parameters andthe partial charges for NMA, TMAO, and water aresummarized in Table 2.All MD simulations were performed at 298 K in a cubic box
of length L. The LJ interactions were spherically truncated at aradius of L/2 and the Ewald method53 was used to treat thelong-range electrostatic interactions. The quaternion formula-tion of the equations of rotational motion was employed, and
Table 1. Overview of Simulations. V, N, and M RepresentBox Volume, Number of Molecules, and MolarConcentration, Respectivelya
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for time integration, we used leapfrog algorithm with a timestep of 10−15 s. Periodic boundary condition and minimumimage convention were used. Initially, the average volume ofthe simulation box corresponding to the desired pressure wasdetermined by performing simulation in isothermal−isobaricensemble (NPT). During this period, the box volume wasallowed to fluctuate. The so obtained box volume was used insubsequent canonical ensemble (NVT) simulation runs. Eachsystem was equilibrated with velocity rescaling to fix thetemperature. Finally, production runs were carried out for 10 nsand these are the results reported.
III. RESULTS AND DISCUSSIONA. Interaction of NMA with Solution Species. As a part
of investigation of NMA interactions with the solutionconstituents, we first computed site−site radial distributionfunctions (rdf) between NMA and different solution species.Selected rdfs that reflect these interactions are shown in Figures1−3.
Figure 1 displays rdfs involving water oxygen (Ow) anddifferent atomic sites in NMA. Focusing on the hydration ofNMA in the absence of osmolyte first, we find that the rdfinvolving carbonyl methyl group (Me1) and water oxygen startsto rise from zero at 3.0 Å, and reaches the value of bulk densityat about 3.4 Å, for which g(r) = 1. Below 3.4 Å, there is actuallyexclusion of water molecules from the solvation shell of thisparticular methyl group. The first minimum, which indicatesthe outer limit of the first hydration shell of Me1, is located at5.4 Å. Although the amide methyl group in NMA (Me2)exhibits similar hydration characteristics, the water distributionis slightly better defined in the vicinity of this group as revealed
Figure 1. NMA−water site−site distribution functions in pure water(black), aqueous urea (red), aqueous TMAO (green), and mixedurea/TMAO (blue) solutions. In mixed osmolyte solution, waterdensity around NMA atomic sites increases dramatically.
Figure 2. NMA−urea site−site distribution functions in aqueous urea(solid) and mixed urea/TMAO (dashed) solutions. Black, red, andgreen lines are for urea carbon (Cu), oxygen (Ou), and nitrogen (Nu),respectively. While O prefers Nu and H prefers Ou, hydrophobicgroups do not show any preference for Ou or Nu. TMAO reduces ureadensity slightly.
Figure 3. NMA−TMAO site−site distribution functions in aqueousTMAO (solid) and mixed urea/TMAO (dashed) solutions. Black, red,and green lines are for TMAO nitrogen (Nt), oxygen (Ot), and carbon(Ct), respectively. TMAO can interact directly with NMA through itsmethyl groups and oxygen atom.
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by an increase in first peak height and an inward movement ofthe first minimum in Me2 − Ow rdf.The carbonyl oxygen and amide hydrogen in NMA are
expected to participate in hydrogen bonding interaction withwater. This particular interaction is demonstrated in rdf profilesof NMA O and H atoms (Figure 1). For pure water, thecontact peaks in O − Hw (not shown) and O − Ow rdf profiles,which characterize the first neighbor, appear at about 1.854 and2.8 Å, respectively, reflecting formation of CO···HwOwhydrogen bonds. Similarly, the H − Ow rdf contains the firstpeak at about 2.0 Å. It is notable that the water density is verylow in the first hydration shell of amide hydrogen. In the centerof the first hydration shell (the first peak), the density of wateris almost equal to the bulk density. The weakness of this peakcan be considered as evidence for much weaker hydration ofthe NMA amide group.Considering the effects of urea and TMAO alone (systems
NU and NT), we see that urea has little effect on distribution ofwater in the vicinity of NMA atomic sites (Figure 1). NMA−water rdfs show only slight awareness also of the presence ofTMAO, except for the H − Ow rdf in which the peakmagnitude increases on addition of TMAO. In contrast tothese, for the system containing both urea and TMAO, there isnoticeable rise of water density around NMA atomic sites.NMA−urea site−site rdfs are shown in Figure 2. Note that
the orientation of urea in the vicinity of the two hydrophobicmethyl groups (Me1 and Me2) is similar to that observed incase of methane and neopentane.55,56 The Nu and Ou rdfprofiles contain first peak at similar positions, indicating thatthere is no preference for urea oxygen or nitrogen near thesehydrophobic groups. The location and height of the first peakin these rdfs also suggest direct interaction of urea with NMAmethyl groups. Again, as expected from a hydrogen bondingperspective between urea and NMA, different orientationalbehaviors of urea molecules are observed near the carbonyloxygen and amide hydrogen in NMA. In particular, the peakpositions (Nu followed by Cu followed by Ou) reflect thepreference of urea nitrogen near the NMA oxygen. On thecontrary, urea oxygen is preferred near the amide hydrogen andurea oxygen accepts this amide hydrogen in NMA−ureahydrogen bonding interaction. From Figure 2, we see that theurea density reduces slightly upon addition of TMAO.Figure 3 exhibits solvation of NMA atomic sites by TMAO.
A side-on orientation of TMAO near the NMA hydrophobicmoieties, as observed previously in cases of methane55 andneopentane,56 can be suggested immediately from the peakpositions. The rdf profiles also indicate a direct interactionbetween methyl groups in NMA and TMAO. Moreover, as inthe case of urea, the amide hydrogen in NMA shows asignificant preference for TMAO oxygen. In the vicinity ofcarbonyl oxygen, a preference of TMAO methyl group can beseen from Figure 3. It is, however, necessary to mention thathydrogen bonding interaction between TMAO methyl hydro-gen and NMA oxygen is unlikely, and although TMAO formshydrogen bonds with NMA hydrogen (see H − Ot rdf), thesmall first peak in H − Nt rdf suggests weak solvation of amidegroup by TMAO. In the center of the first solvation shell ofNMA hydrogen, density of TMAO is about 0.7 times the bulkdensity. Slightly weaker NMA-TMAO peaks are observed inurea/TMAO mixture.Further insight into the solvation of NMA was obtained by
computing the average number of water and osmolytemolecules (central atom only) around the NMA atomic sites.
The average number of molecules was calculated from thecorresponding rdf, gαβ, using the relation
∫πρ=αβ β αβn r g r r4 ( ) dr
0
2c
(2)
where nαβ represents the number of atoms of type βsurrounding atom α in a shell extending from 0 to rc and ρβis the number density of β in the system.The running coordination numbers for different atomic sites
in NMA are shown in Figure 4. Because urea and TMAO are
significantly larger in size than water, exclusion of these twoosmolytes from a certain volume shell around the atomic sitesof NMA is expected for purely geometric reasons, and this isreflected in larger radii of exclusion for these two osmolytes.The relatively closer approach of urea as compared to TMAOcan be interpreted as better contact of NMA with urea thanwith TMAO. The point that also emerges from Figure 4 is thereplacement of a large number of water molecules in thesolvation shell of each NMA atomic site by osmolytes. Thetotal number of water molecules in the solvation shell of NMAmolecule (shown in Figure 5) is much higher in pure waterthan in aqueous solution of osmolyte. Considering the effect ofurea alone, i.e., in the absence of TMAO, we see that, in thesolvation shell of radius 6 Å, NMA loses about 30 watermolecules, and about 16 urea molecules enter in this particularregion. This corresponds to replacement of about 2 hydrationwater by each urea molecule. Due to this higher exclusion ofwater, total number of solution species (water plus urea) in theNMA solvation shell is much higher in pure water than in ureasolution (Figure 5).
Figure 4. Running coordination numbers for different NMA atomicsites in pure water (black), aqueous urea (red), aqueous TMAO(green), and mixed urea/TMAO (blue) solutions. Solid, dashed, anddotted lines are for water, urea, and TMAO, respectively. Both ureaand TMAO replace water molecules that exist in the solvation shell ofNMA atomic sites.
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For the simulation of NMA in aqueous TMAO solution(system NT), we find that the existence of TMAO in thesolvation shell leads to reduction in hydration water (Figure 5).To be specific, each TMAO molecule can replace about 2.5hydration water of NMA. However, since the number ofTMAO molecules that enter in NMA solvation shell issignificantly small (about 7 in contrast to about 16 for urea),NMA dehydration is lower in aqueous solution of TMAO incomparison to that of urea.Comparison of the solvation number of NMA in urea/
TMAO mixture with that in binary urea solution revealsremoval of both water and urea from the solvation shell ofNMA. Nonetheless, removal of urea is much less pronouncedas compared to water.To describe the difference between the local environment of
NMA and that of the bulk solution, we then calculated apreferential binding parameter. Following our previous work,54
we defined the binding parameter, τi/j, as
τ = −rn rn r
NN
( )( )( )i j
i
j
i
j/
(3)
where n(r) is the number of molecules (central atom only) at aparticular distance around NMA atomic sites and N representsthe total number of molecules in the system with subscripts iand j standing for species i and j, respectively. This bindingparameter measures the local ratio of species i to j minus theirbulk ratio and, hence, gives deviation from an ideal solvationmodel. A negative value of τi/j indicates preferentialaccumulation of j over i and its positive value indicatespreferential exclusion of j in that region. Note that, while theglobal thermodynamic preferential interaction parameters arerelated to transfer chemical potentials (see, e.g., works by the
solvation experts Schellman, Timasheff, or Parsegian), we arenot attempting to correlate our τi/j values to transfer chemicalpotentials. The parameter used here gives only a qualitative ideaof exclusion or accumulation as a function of distance.Figure 6 shows the τw/u and τw/t values for different atomic
sites in NMA as a function of distance. The regions of
preferential exclusion of urea and TMAO are clearly visible inthis figure. The initial positive value of the parameter is directlyrelated to the larger radii of exclusion (the longest distance forwhich g(r) = 0) for urea carbon and TMAO nitrogen. Therelative distribution of water to osmolyte is more ideal like inthe case of urea. Thus, NMA atomic sites do not show anypreference for water and urea, and these solution species aredistributed uniformly. TMAO, on the other hand, dominantlyoccupies some specific regions and the relative distribution isnonuniform. The first valleys in TMAO binding parameters arerelated to TMAO interactions with NMA atomic sites throughits methyl groups and oxygen atom. The slight inwardmovement of the radius of preferential accumulation (shortestdistance for which τi/j has minimum value) for urea ascompared to that of TMAO suggests better contact of NMAwith urea than with TMAO. Remarkably, addition of a secondosmolyte makes the solvation layer of NMA better mixed interms of solution species.For further characterization of NMA solvation, we
investigated the structural, energetic, and dynamical propertiesof hydrogen bonds formed by NMA with solution species. Themethods and the geometric criteria for hydrogen bondproperties and dynamics calculations were identical to thosein our recently published work.54 In short, a hydrogen bondwas defined by imposing cutoff distances of rDA for D − A(where D is donor and A is acceptor) and rAH for A − H, and asimultaneous cutoff angle of 45° for H − D − A. We selectedcutoff distances according to the position of the first minimumof the appropriate rdf and these distances are tabulated in Table3. We considered the five possible hydrogen bonds between
Figure 5. Top: Total number of water (solid), urea (dashed), andTMAO (dotted) molecules in the solvation shell of NMA as a functionof distance. Bottom: Total number of solution species in the solvationshell of NMA as a function of distance. Color codes are as in Figure 1. Figure 6. Preferential interaction parameter for NMA atomic sites in
aqueous urea (red), aqueous TMAO (green), and mixed urea/TMAO(blue) solutions. Better contact of NMA with urea than with TMAOcan be interpreted from lower radius of preferential accumulation.
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NMA and solution species: O···HwOw, Ow···HN, O···HuNu,Ou···HN, and Ot···HN. Note that hydrogen bonds in this studyare abbreviated as Im−Jn where I and J can be N (for NMA),W (for water), U (for urea), and T (for TMAO) and m and ncan be a (for acceptor) and d (for donor). Further, hydrogenbonds are with respect to the first species. For example, theO···HwOw bond is abbreviated as Na − Wd and the hydrogenbond number is with respect to NMA oxygen.To investigate hydrogen bond dynamics, we defined a
continuous hydrogen bond correlation function, SHB, as
=S t h H t h( ) (0) ( ) /HB (4)
where h and H are two hydrogen bond population variables. If aparticular tagged pair of particles is hydrogen bonded at time t,h is unity, and is zero otherwise. In the case of H, if the taggedpair of particles remains continuously hydrogen bonded frominitial period to time t, it takes a value of unity, and is zerootherwise. The lifetime of a hydrogen bond, τHB, can beobtained by time integration of SHB.In Table 4, we have presented the total (considering all 20
NMA molecules) number of hydrogen bonds formed betweenNMA and solution species together with energies of thesebonds. Figure 7, on the other hand, illustrates the fraction ofNMA molecules that engage in n number of hydrogen bondswith solution species. In Figure 7, we find that most of thecarbonyl oxygens participate in 2 hydrogen bonds with water,although some of these are also 1 or 3 coordinated to water. Onaverage, each NMA oxygen is found to have about 1.7hydrogen bonds to water. The amide hydrogen remainssatisfied by forming a single hydrogen bond to water moleculeand the average number of Nd − Wa hydrogen bonds perNMA is only 0.75. Thus, hydrogen bonding between NMA andwater is solely dominated by carbonyl oxygen in NMA, which isnot surprising. Table 4 shows that, out of the total 49 NMA−water hydrogen bonds in pure water, 34 go to carbonyl oxygen,leaving only 15 for amide hydrogen.In binary solution of urea, NMA molecules lose significant
number of hydrogen bonds to water (Table 4) and almost all ofthese hydrogen bonds are replaced by NMA−urea hydrogen
bonds, keeping the total number of hydrogen bonds betweenNMA and solution species close to pure water system. Notethat the effect of urea on the NMA−water hydrogen bondnumber is dramatic for both amide hydrogen and carbonyloxygen. In most cases, the two solvation sites of carbonyloxygen are shared by one water molecule and one ureamolecule (Figure 7). Although there are some NMA oxygensthat participate in 2 hydrogen bonds with water even in thepresence of urea, the number is significantly lower than that in
Table 3. Cutoff Distances (in Å) for the Hydrogen Bonds Investigateda
aN, W, U, and T represent NMA, water, urea, and TMAO, respectively. The terms “Donor” and “Acceptor” in the first column are, respectively, forhydrogen bonds with first species behaving as donor and acceptor. rDA and rAH are defined in the text.
Table 4. Properties of Hydrogen Bonds Formed by NMA with Water and Osmolytea
anHB and EHB represent total number of hydrogen bonds and energy (in kJ mol−1) per hydrogen bond, respectively. Hydrogen bond types aredefined in the text.
Figure 7. Fraction of NMA molecules that engage in n number ofhydrogen bonds with water and osmolyte in pure water (black),aqueous urea (red), aqueous TMAO (green), and mixed urea/TMAO(blue) solutions. Hydrogen bond types are defined in the text and thenumbers are with respect to the first species. Considering the fact thatTMAO cannot form hydrogen bonds with NMA oxygen, reduction inrelative population of NMA oxygen that participate in 2 hydrogenbonds with water in the presence of TMAO is striking and likely themost significant.
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the case of pure water. The average number of Na − Wdhydrogen bonds per NMA reduces from about 1.7 in purewater to about 1.1 in binary urea solution and there arises about0.5 Na − Ud hydrogen bonds. What these results support is thedirect interaction model for urea-conferred protein denatura-tion.8−22 Urea removes water from the protein solvation shelland makes some favorable contact with protein residues,leading to protein unfolding.Just like in the case of urea, addition of TMAO reduces the
number of Nd − Wa hydrogen bonds. The loss is, however,very small and is also well compensated by formation of few Nd− Ta hydrogen bonds (Table 4). We observe that the averagenumber of Na − Wd bonds decreases dramatically uponaddition of TMAO. With the fact that TMAO cannot formhydrogen bonds with carbonyl oxygen in NMA taken intoconsideration, the so-observed large reduction in Na − Wdhydrogen bonds is striking and likely the most significant.Figure 7 shows that the relative population of carbonyl oxygenthat participate in 2 hydrogen bonds with water is reduced inbinary TMAO solution, whereas that participating in 1hydrogen bond increases. Since TMAO is unable tocompensate the loss of Na − Wd hydrogen bonds, the totalnumber of NMA−solvent hydrogen bonds reduces from about49 in pure water to about 43 in binary TMAO solution (Table4).It is interesting to observe that TMAO does not affect the
number of NMA−urea hydrogen bonds and there is almost nochange in the relative population of NMA molecules thatparticipate in 1 and 2 hydrogen bonds with urea (Figure 7).This means that prevention of protein−urea hydrogen bondformation by TMAO cannot be considered as a primary reasonfor counteracting the protein denaturing effect of urea. On theother hand, the average number of NMA−water hydrogenbonds is lower in the urea/TMAO mixture as compared tobinary urea solution (Table 4), raising the possibility thatTMAO counteracts urea denaturation of proteins by preventingformation of protein−water hydrogen bonds. Remarkably, MDsimulations of the protein chymotrypsin inhibitor 2 (CI2) byBennion and Daggett57 suggested that TMAO does notstabilize the native state by occluding urea from the solvationshell. Similarly, Kokubo et al.43 did not observe disturbance ofpeptide−urea hydrogen bonds in urea/TMAO mixture. Incontrast, these studies showed dehydration of protein and asignificant loss of peptide−water hydrogen bonds uponaddition of TMAO.43,57 Again, from Figure 7, it can be seenthat there is not much difference in relative population ofhydrogen bonds for Nd − Wa between binary and ternarysolutions of urea. The largest differences arise only from adecrease in relative population of carbonyl oxygen thatparticipate in 2 hydrogen bonds to water. On these grounds,it is reasonable to suggest here that the unavailability of waterto form hydrogen bonds with backbone oxygen and TMAO’sinability to compensate the loss of these hydrogen bonds arethe most likely reasons for protein stabilizing and counteractingability of TMAO.In Table 4, we find that Na−Wd hydrogen bond is weaker
than the Na − Ud bond by about 2 kJ/mol. The energydistribution curve is much broader for the latter, with someenergetically strong hydrogen bonds (Figure 8) and also thehydrogen bond relaxes slowly as compared to Na −Wd both inbinary and ternary solutions of urea (Figure 9). Note that therelative population of high energy (more negative) bondsslightly increases for Na − Wd and decreases for Na − Ud
Figure 8. Probability distribution of hydrogen bond energies foroxygen (left panel) and hydrogen (right panel) of NMA with water(top) and urea (bottom) in pure water (black), aqueous urea (red),aqueous TMAO (green), and mixed urea/TMAO (blue) solutions.Dashed lines in bottom-right panel are for TMAO-NMA hydrogenbonds.
Figure 9. Time dependence of the continuous hydrogen bondcorrelation functions, SHB, for oxygen (left panel) and hydrogen (rightpanel) of NMA with water (top) and urea (bottom) in pure water(black), aqueous urea (red), aqueous TMAO (green), and mixedurea/TMAO (blue) solutions. Dashed lines in bottom-right panel arefor TMAO-NMA hydrogen bonds.
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upon addition of TMAO. The Nd − Ta hydrogen bond is thestrongest among the hydrogen bonds investigated here. For theurea/TMAO mixture, the Nd − Ta bond is more attractivethan the Nd − Ua bond by about 7 kJ/mol which, in turn, isabout 9 kJ/mol more attractive than the Nd − Wa hydrogenbond. As revealed in Figures 8 and 9, on moving from Nd −Wa through Nd − Ua to Nd − Ta, the energy distributioncurve shifts to more attractive region and the hydrogen bondrelaxation become slower and slower. We note that magnitudesof these hydrogen bond energies do not change significantly onchanging the system.Thus, a thorough investigation of the solvation characteristics
of NMA atomic sites in pure water and in binary and ternarysolutions of urea and TMAO reveals replacement of a largenumber of NMA hydration water by these two osmolytes.While both urea and TMAO are found to interact directly withNMA atomic sites, NMA can make some better contact withurea than with TMAO. NMA loses some hydrogen bonds towater due to the inclusion of osmolytes in its solvation shell.The hydrogen bonds of NMA oxygen with waters becomestabilized in the presence of TMAO, but the relativestabilization is not large and this energetic gain can be expectedto overcome easily by the energetic cost due to the loss of largenumber of hydrogen bonds to water. Urea reduces the loss ofNMA-water hydrogen bonds by forming hydrogen bonds withNMA hydrogen bonding sites, whereas TMAO cannot formhydrogen bonds with NMA oxygen and hence shows itsinability to compensate the loss of hydrogen bonds betweenNMA and water. What these findings suggest is that, unlikeurea, which can stabilize unfolded protein by forming hydrogenbonds with its backbone, TMAO prevents backbone oxygenfrom interacting efficiently with solution species, providing thepathway for TMAO-induced protein stabilization and counter-action. Two other possible pathways to investigate theunderlying mechanism by which TMAO stabilizes proteinand counteracts protein denaturation by urea are to examinethe solvation of TMAO by water and urea (which eventuallyreduces availability of water and urea to solvate proteinresidues) and also the indirect effect of TMAO on waterstructure. In the following, we have discussed both of these twopossibilities in aqueous NMA solutions at different environ-ments.B. Interactions between Solution Species. In an
attempt to understand the origin of the counteracting effectof TMAO, we studied the solvation of both hydrophobic(methyl group) and hydrogen bonding (oxygen atom) sites ofTMAO by water and urea and also examined the hydrogenbonding interactions between the solution components. Figure10 displays selected rdfs that show the solvation of TMAO bywater and urea. We can see immediately that the first peak in Ot− Ow rdf is much stronger than that in Ct − Ow rdf,demonstrating strong affinity of TMAO for water comingthrough TMAO−water hydrogen bonding. Urea has little effecton the hydration shell of methyl group, but increases the firstpeak in Ot − Ow rdf dramatically. So, the water moleculesbecome tightly coordinated to TMAO oxygen upon addition ofurea. However, the number of water molecules in the firsthydration shell of TMAO methyl group decreases in urea/TMAO mixture (Table 5). Similarly, TMAO oxygen losesabout 0.7 first shell water molecules in the presence of urea,ultimately leading to overall loss of about 8 water molecules inthe solvation shell of TMAO.
It is striking to note that the general features of the Ct − Cuand Ot − Nu functions are similar to those of the Ct − Ow andOt − Ow rdfs. The qualitative similarity of these rdf pairsindicates that urea can solvate both hydrophobic and hydrogenbonding sites of TMAO, as do water molecules. The density ofurea near the TMAO methyl group is very similar to that ofwater, suggesting no preference of hydrophobic sites for waterand urea. However, as reflected by the much stronger first peakin Ot − Ow rdf than that in Ot − Nu, water interacts more withTMAO than does urea.In Figure 11, we have shown rdfs for atom pairs associated
with hydrogen bonding. The following observations are madefrom this figure: (a) The first peak in Ou − Hw rdf is muchstronger than that in Hu − Ow, indicating that urea prefers to bea hydrogen bond acceptor rather than a donor in urea−waterhydrogen bonds. (b) Relative to water hydrogen, first peaks areshifted outward for urea hydrogen. (c) Although both waterand urea hydrogens are tightly coordinated to TMAO oxygen,the tendency of TMAO oxygen for water hydrogen is muchhigher.
Figure 10. Selected site−site rdfs that show solvation of TMAO bywater and urea in aqueous TMAO (green) and mixed urea/TMAO(blue) solutions. Urea can solvate both hydrophobic and hydrogenbonding sites of TMAO as do water molecules.
Table 5. Coordination Numbers for TMAO Atomic Sitesa
atom pair rc (Å) NT NUT
Ct − Ow 4.4 7.0 4.5Ot − Ow 3.4 2.7 2.0Nt − Ow 6.3 23.5 15.1Ct − Cu 5.0 2.0Ot − Nu 3.8 1.2Nt − Cu 6.4 4.7
arc is the integration distance. Numbers are with respect to first atomicsite. For Nu, the actual number is multiplied by 2.0 to consider bothnitrogen atoms in urea.
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To obtain further insight into the nature of hydrogenbonding interactions, we computed the strength and number ofthe hydrogen bonds by adopting the geometric criteria definedabove. The cutoff distances for the hydrogen bonds are listed inTable 3.Figure 12 illustrates the fraction of molecules ( f n) that
engage in n number of hydrogen bonds, and the averagenumber of hydrogen bonds per molecule are depicted in Table6. For pure water, most of the water molecules participate ineither 3 or 4 hydrogen bonds. Small fractions of watermolecules are engaged in either 2 or 5 hydrogen bonds. Theeffect of osmolyte on water hydrogen bonding network isdramatic. It reduces the number of higher (4 and 5)coordinated water molecules and, at the same time, increasesthe number of lower (1−3) coordinated water molecules. As aresult, the average number of water−water hydrogen bonds isreduced in aqueous solutions of osmolyte (Table 6). Note thatthe changes are significantly pronounced for 2- and 4-coordinated water molecules and are related to the numberof osmolyte molecules that have to be accommodated in thecavities of water molecules.In binary solution, the majority of the urea oxygens are found
to have either 1 or 2 hydrogen bonds to water (Figure 12) and,on average, there are about 2.6 water molecules which arehydrogen bonded to each urea molecule (Table 6). As revealedin Figure 12, although self-association of urea moleculesthrough hydrogen bonding is possible, urea prefers to formthe maximum number of hydrogen bonds with watermolecules. The average number of urea−urea hydrogenbonds is significantly smaller than that of urea−water hydrogenbonds per urea molecule. Addition of TMAO to the systemincreases the relative population of 1-coordinated watermolecule and decreases those of 2- and 3-coordinated watermolecules for oxygen of urea. The relative population of 1-coordinated water molecule to urea hydrogen remains similarin the ternary solution, whereas the fraction of 2- and 3-
coordinated water molecules decreases. This ultimately leads toa reduction of about 0.3 urea−water hydrogen bonds in theurea/TMAO mixture. TMAO also breaks some urea−ureahydrogen bonds, and so, although urea forms some hydrogenbonds with TMAO molecule (about 0.2 per urea), the totalnumber of hydrogen bonds per urea molecule is reduced in theternary osmolyte solution. These observations are consistentwith a direct interaction between urea and TMAO. In theabsence of TMAO, urea hydrogens are free to interact withoxygens of water and urea. As TMAO is added, a fraction ofthese hydrogen bonds are replaced by TMAO−urea hydrogenbonds.In contrast to urea, the majority of TMAO molecules
participate in 3 hydrogen bonds with water (Figure 12). Thenumber of TMAO molecules engaged in 2 hydrogen bondswith water is also significant and, on average, there are about2.5 water molecules that are found to be hydrogen bonded toTMAO oxygen. Upon addition of urea, the third hydrogenbond of TMAO oxygen to water is likely to be replaced by anew hydrogen bond between TMAO and urea. Each TMAOmolecule now forms hydrogen bonds mostly with 2 watermolecules and, on average, the total number of hydrogen bondsper TMAO molecule (2.5) remains similar to that of its binarysolution.The average energies and lifetimes of hydrogen bonds
between these solution species are given in Table 6. Figures 13and 14, on the other hand, show the probability distributions ofthe hydrogen bond energies and time dependence of thehydrogen bond correlation functions. These results lead toseveral interesting observations. We see that urea has anegligible effect on water−water hydrogen bond energy. Thechange in energy distribution is very small in binary ureasolution. The water−water hydrogen bond relaxes slowly in the
Figure 11. Selected site−site rdfs showing hydrogen bondinginteractions between solution species in pure water (black), aqueousurea (red), aqueous TMAO (green), and mixed urea/TMAO (blue)solutions. Solid and dashed lines are for water and urea hydrogens,respectively.
Figure 12. Fraction of molecules with n number of hydrogen bondsbetween solution species in pure water (black), aqueous urea (red),aqueous TMAO (green), and mixed urea/TMAO (blue) solutions.Hydrogen bond types are defined in the text and the numbers are withrespect to the first species. The solvation sites of TMAO oxygen, thatare occupied by 2 to 3 water molecules in binary TMAO solution,become available to both water and urea upon addition of urea,reducing the number of TMAO molecules that engage in 3 hydrogenbonds with water.
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presence of urea (as compared to pure water), and the lifeexpectancy of this hydrogen bond increases slightly (from 1.25ps in pure water to 1.41 ps in binary urea solution). Theseresults do not provide any evidence for urea’s ability to disturbwater hydrogen bonding network. In fact, previous simulationsshowed enhancement of the first peak in Ow − Ow rdf,indicating a tightly coordinated first shell of water, and only aslight collapse of the water second shell was noted.23,25,55,56
The effect of TMAO on water hydrogen bonding interactionis more pronounced. In particular, hydrogen bonds becomemore attractive (Table 6) and the energy distribution shiftstoward the higher energy (more negative) side in aqueoussolutions of TMAO, indicating water hydrogen bond networkstabilization by TMAO. Water−water hydrogen bond relaxa-tion is also much slower in the presence of TMAO, and thehydrogen bond lifetime increases to 1.77 ps in binary TMAOsolution. It is notable here that TMAO’s ability to stabilize thehydrogen bonding network of water has been suggested to be
an important factor to counter the protein denaturing effect ofurea.57
Considering the case of urea−water interaction, we find thatthe Ua − Wd bond is more attractive than the Ud − Wahydrogen bond and, in fact, the average energy (≈ −15 kJ/mol) for the latter is the lowest among the hydrogen bondsinvestigated in this study. For hydrogen bonds formed by ureaoxygen with water hydrogen, energy distribution is very similarto that for water−water hydrogen bonds. In contrast, thedistribution is broader for hydrogen bonds between ureahydrogen and water oxygen and the population of low-energyhydrogen bonds is significantly higher in this case. There is,however, no such differences between these two hydrogenbonds in terms of their relaxation with time, and both havesimilar hydrogen bond lifetimes (about 0.9 ps in binary ureasolution). Although there is negligible change of urea−waterhydrogen bond energies in the presence of TMAO, relaxation
Table 6. Properties of Hydrogen Bonds Involving Water and Osmolytea
NW NU NT NUT
Bond type nHB (EHB) τHB nHB (EHB) τHB nHB (EHB) τHB nHB (EHB) τHB
W − W 3.52 1.25 2.84 1.41 3.20 1.77 2.51 1.98(−19.17) (−19.26) (−19.73) (−19.85)
Wa − Ud .... .... 0.32 0.91 .... .... 0.33 1.14(....) (−15.09) ...... (−15.25)
Ta − Ud .... .... .... .... .... .... 0.42 2.65(....) (....) ...... (−29.59)
anHB, EHB, and τHB represent average number, energy (in kJ mol−1), and lifetime (in ps), respectively. Hydrogen bond types are defined in the textand hydrogen bond numbers are with respect to the first species.
Figure 13. Probability distribution of hydrogen bond energies foroxygen atoms of water, urea, and TMAO (from top to bottom,respectively) in pure water (black), aqueous urea (red), aqueousTMAO (green), and mixed urea/TMAO (blue) solutions. Solid anddashed lines are for water and urea hydrogens.
Figure 14. Time dependence of the correlation functions, SHB forhydrogen bonds involving oxygen atoms of water, urea, and TMAO(from top to bottom, respectively) in pure water (black), aqueous urea(red), aqueous TMAO (green), and mixed urea/TMAO (blue)solutions. Solid and dashed lines are for water and urea hydrogens.
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of urea−water hydrogen bond becomes slower as TMAO isadded to the system.The hydrogen bonds formed by TMAO oxygen with both
water and urea are energetically very strong (about −29 kJ/mol). For the ternary solution, the TMAO−water hydrogenbond is more attractive than the water−water and urea−water(urea acceptor) hydrogen bonds by about 9 kJ/mol. Similarly,the TMAO−urea hydrogen bond is about 5 kJ/mol moreattractive than the urea−urea bond. The energy distributionsshow a large population of these hydrogen bonds in highenergy (more negative) regions. What is more, these hydrogenbonds decay very slowly in aqueous solutions. The averagelifetime of the TMAO−water hydrogen bond is found to be3.92 ps in the urea/TMAO mixture, which is about 2 timeshigher than that between water molecules. We note thatsignificantly longer hydrogen bond lifetime for a TMAO−waterthan for a water−water hydrogen bond has been widelyreported in the literature.36,37,58−60 The MD simulations alsopredicted that TMAO−water hydrogen bonds are moreattractive than water−water hydrogen bonds.37
Hence, examining the solvation characteristics of TMAO, wefind that both water and urea can solvate TMAO molecules,and due to the existence of urea in its solvation shell, TMAOloses some hydration waters upon addition of urea. Solvation ofTMAO by water, on the other hand, leads to relativestabilization of water hydrogen bonding network. Theseobservations correspond well with previously observedsolvation of TMAO by water and urea37,61,62 and also withthe water structure making ability of TMAO.23,25,36
IV. SUMMARY AND CONCLUSIONSTo elucidate the mechanism of TMAO-induced counteractionof urea-conferred protein denaturation, we investigatedsolvation characteristics of NMA in binary and ternary solutionsof urea and TMAO. Computations of site−site rdfs indicateddirect interactions of both urea and TMAO with NMA atomicsites. It was evident from analysis of solvation number that thenumber of TMAO molecules that enter the NMA solvationshell was significantly lower than that of urea, and due to theinclusion of osmolytes, NMA molecules were dehydrated inbinary and ternary solutions of urea and TMAO. Bothosmolytes were observed to replace a large number of watermolecules in the solvation shell of each NMA atomic site,though the water removing ability of TMAO was slightly higherthan that of urea (2.5 per TMAO against 2.0 per urea).Addition of a second osmolyte also reduced the number of theother osmolyte in the solvation shell, but the effect was muchless pronounced in comparison to that of hydration water. Thelocal environment of NMA was further explored by calculatingthe preferential binding parameter, and it was observed thaturea made better contact with NMA than did TMAO and thesolvation layer of NMA was better mixed in terms of water andurea as compared to that of water and TMAO.Investigation of structural, energetic, and dynamical proper-
ties of NMA hydrogen bonds with solution species showed thathydrogen bonding between NMA and water was largelydominated by carbonyl oxygen in NMA. Our resultsdemonstrated the replacement of a large number of NMA−water hydrogen bonds by NMA−urea hydrogen bonds and thetotal number of hydrogen bonds between NMA and solutionspecies was found to be similar in pure water and in binary ureasolution. The two solvation sites in NMA oxygen (which wereoccupied by water alone in pure water) were shared by water
and urea in aqueous urea solution. Hydrogen bonds betweenNMA and urea were found to be more attractive than thosebetween NMA and water and the relaxation was also slower forthe former. On the other hand, our analysis of hydrogen bondproperties did not provide any evidence for the “water structurebreaking” capacity of urea. Rather, urea was found to slightlyincrease water−water hydrogen bond energy and lifetime. All ofour results are consistent with a direct interaction model forurea-conferred protein denaturation, but do not support theindirect mechanism that presumes urea as a structure breakerfor water, making water molecules free to interact with proteinresidues.Just like urea, TMAO was observed to replace the hydrogen
bond between amide hydrogen and water (though the changewas negligible), but the most striking observation was thereduction in hydrogen bonds between carbonyl oxygen andwater upon addition of TMAO. Note that TMAO cannotdonate its hydrogen to NMA oxygen and hence was unable tocompensate for the loss of NMA−water hydrogen bonds.There was no indication of a decrease in NMA−urea hydrogenbond numbers caused by the addition of TMAO, but relative tobinary urea solution, NMA lost some hydrogen bonds to watermolecules. Furthermore, TMAO showed its effect mostly onthe hydrogen bonding capacity of NMA oxygen. These lead usto suggest that it is not the effect of TMAO on protein−ureahydrogen bonding interaction that reduces urea’s deleteriouseffect on protein. Rather, the native structure of protein ismaintained in the urea/TMAO mixture due to the unavail-ability of water to form hydrogen bonds with backbone oxygenand TMAO’s inability to compensate the loss of thesehydrogen bonds.To shed light on the counteracting mechanism, we then
examined solvation of TMAO by water and urea and alsostudied structural, energetic, and dynamical properties ofvarious possible hydrogen bonds between solution species.Urea was seen to solvate both hydrophobic and hydrogenbonding sites in TMAO, and because of its interaction withurea, TMAO lost some hydration waters upon addition of urea.TMAO oxygen, which participated in 2 to 3 hydrogen bondswith water in its binary solution, showed its inability tomaintain these hydrogen bonds as urea was added and thesehydrogen bonds were replaced by TMAO−urea hydrogenbonds. We observed that hydrogen bonds formed by TMAOwith water and urea were very strong. In fact, these hydrogenbonds were found to be energetically much stronger than thosebetween water and urea and also had high lifetimes. In additionto its direct interaction with water and urea, TMAO alsoshowed an indirect effect on water−water and urea−waterinteraction in which it stabilized the water hydrogen bondingnetwork and also reduced relaxation of urea−water hydrogenbonds. These results are consistent with previously observedsolvation of TMAO by water and urea37,61,62 and also with thewater structure making ability of TMAO.23,25,36 Note that,although water structure enhancement by TMAO is consistentwith NMA dehydration in the presence of TMAO and also withthe loss of NMA−water hydrogen bonds, by no means can thewater structure making property of TMAO be considered to bea primary factor in protecting proteins in presence of urea. Theindirect effect of TMAO on water structure was observedpreviously to be correlated with hydration of a carbon nanotubeinterior, but at the same time, the authors reported that theeffect was associated strongly with its direct interaction.25
Similarly, Athawale et al. mentioned the limitations of
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arguments that relate enhanced or decreased water structure tomacromolecular thermodynamics.46 Again, solvation of TMAOby both water and urea is consistent with the proposedpreferential solvation mechanism37,61 of TMAO counteraction,whereby both water and urea prefer to solvate TMAO ratherthan the protein, and corresponding to this model, loss ofNMA−water hydrogen bonds were observed in both binaryand ternary solutions of TMAO. However, the same was notapplicable in the case of urea, for which there was no suchdecrease in hydrogen bond number. Similar observations weremade recently by Kokubo et al.43 and are also consistent withthe experimental findings that the efficiency of an osmolyte toact on the protein is not interfered by the presence of a secondosmolyte.44,45 Thus, it is also difficult to infer the mechanism ofTMAO counteraction of urea-induced protein denaturation bythe preferential solvation model alone.Then, what could be the most likely reason that makes
TMAO an effective protein stabilizer and counteractant of ureadenaturation? In our opinion, it is the inefficient interaction ofTMAO with protein backbone that is responsible in thisprocess. Although TMAO has some tendency to solvatenonpolar sites in proteins and also interacts directly withbackbone NH group and positively charged side chains,63 theseinteractions are not sufficient to stabilize the unfolded state ofprotein. More importantly, unlike urea, TMAO cannot donateits hydrogen to backbone oxygen and hence shows its inabilityto compensate for the loss of a significant number of hydrogenbonds between backbone oxygen and water that is caused byinclusion of a few TMAO molecules in the solvation shell of theprotein (despite the preferential exclusion of TMAO). Thus,the total number of favorable contacts of protein with solutionspecies is reduced upon the addition of TMAO (as comparedto pure water). TMAO induces protein folding because thecompact state has more energetically favorable interactions(intraprotein plus few protein−solvent) than the extended state(only protein−solvent). What is more, folding of protein inaqueous TMAO solution allows TMAO to maximize thenumber of favorable contacts with water in the bulk and also tostabilize the water hydrogen bonding network. The picturedoes not change even in the urea/TMAO mixture. Ascompared to binary urea solution, in which the protein prefersits solvent-exposed state to maximize favorable contacts withsolution species, the number of efficient interaction sites for theprotein is reduced as TMAO is added. Since the relativestability of the unfolded state gained by protein−solventinteractions is overcome by various intraprotein plus fewprotein−solvent interactions in the folded state, the proteinadopts a (folded) structure in ternary solutions of urea andTMAO that is similar to that in pure water. The only differenceis that some urea molecules now appear in close proximity ofthe protein. The highly favorable interactions of TMAO withwater and urea in the bulk give further stability to the systemthat contains the folded protein. Hence, in all likelihood, theinefficient interaction of TMAO with protein backbone playsthe most important role in the ability of TMAO to stabilizeproteins and offset protein denaturation by urea. A note ofcaution, however, is that, although hydrogen bond numberprovides a useful measure to describe protein stability, there areseveral other factors effective in protein stabilization. Asystematic study with proteins will be needed to get to theconclusion of the counteracting mechanism.
■ ACKNOWLEDGMENTSThe financial support of the Council of Scientific and IndustrialResearch (CSIR) and Department of Science and Technology(DST), Govt. of India are gratefully acknowledged.
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