The effect of aqueous solutions of trimethylamine-N-oxide on pressure induced modifications of...

The effect of aqueous solutions of trimethylamine-N-oxide on pressureinduced modifications of hydrophobic interactionsRahul Sarma and Sandip Paul Citation J Chem Phys 137 094502 (2012) doi 10106314748101 View online httpdxdoiorg10106314748101 View Table of Contents httpjcpaiporgresource1JCPSA6v137i9 Published by the American Institute of Physics Additional information on J Chem PhysJournal Homepage httpjcpaiporg Journal Information httpjcpaiporgaboutabout_the_journal Top downloads httpjcpaiporgfeaturesmost_downloaded Information for Authors httpjcpaiporgauthors

Downloaded 02 Oct 2012 to 1716734205 Redistribution subject to AIP license or copyright see httpjcpaiporgaboutrights_and_permissions

THE JOURNAL OF CHEMICAL PHYSICS 137 094502 (2012)

The effect of aqueous solutions of trimethylamine-N-oxide on pressureinduced modifications of hydrophobic interactions

Rahul Sarma and Sandip Paula)

Department of Chemistry Indian Institute of Technology Guwahati Assam 781039 India

(Received 28 May 2012 accepted 13 August 2012 published online 4 September 2012)

To understand the mechanism of protein protection by the osmolyte trimethylamine-N-oxide(TMAO) at high pressure using molecular dynamics (MD) simulations solvation of hydrophobicgroup is probed in aqueous solutions of TMAO over a wide range of pressures relevant to proteindenaturation The hydrophobic solute considered in this study is neopentane which is a consider-ably large molecule The concentrations of TMAO range from 0 to 4 M and for each TMAO con-centration simulations are performed at five different pressures ranging from 1 atm to 8000 atmPotentials of mean force are calculated and the relative stability of solvent-separated state over theassociated state of hydrophobic solute are estimated Results suggest that high pressure reduces asso-ciation of hydrophobic solutes From computations of site-site radial distribution function followedby analysis of coordination number it is found that water molecules are tightly packed around thenonpolar particle at high pressure and the hydration number increases with increasing pressure Onthe other hand neopentane interacts preferentially with TMAO over water and although hydrationof neopentane reduces in presence of this osmolyte TMAO does not show any tendency to preventthe pressure-induced dispersion of neopentane moieties It is also observed that TMAO moleculesprefer a side-on orientation near the neopentane surface allowing its oxygen atom to form favorablehydrogen bonds with water while maintaining some hydrophobic contacts with neopentane Analy-sis of hydrogen-bond properties and solvation characteristics of TMAO reveals that TMAO can formhydrogen bonds with water and it reduces the identical nearest neighbor water molecules caused byhigh hydrostatic pressures Moreover TMAO enhances life-time of waterndashwater hydrogen bonds andmakes these hydrogen bonds more attractive Implication of these results for counteracting effect ofTMAO against protein denaturation at high pressures are discussed copy 2012 American Institute ofPhysics [httpdxdoiorg10106314748101]

I INTRODUCTION

It is well known that the stability of native proteins inaqueous solution depends strongly on the thermodynamicvariables such as pressure1ndash19 as well as on the surround-ing chemical environments20ndash25 and in recent years effectof chemical agents on protein structure and stability asso-ciated with pressure denaturation has received increasinginterest26ndash37 Different co-solvents have well-documented ef-fects on protein stability at high pressure Chaotropic agentssuch as urea make protein to unfold at a lower pressure ascompared to the pure water system26 In contrast polyhy-dric alcohols (glycerol sorbitol) can increase the denatura-tion pressure26 It has been reported that the concentrationof a naturally occurring osmolyte trimethylamine-N-oxide(TMAO) increases linearly with depth of the sea both amongdifferent species and within the same species while reduc-ing the concentration of the common osmolytes of shallowrelatives eg glycine in shrimp urea in skates27ndash30 Thisparticular osmolyte (TMAO) was found to offset pressure-inhibition of stability of several lactate dehydrogenase ho-mologues polymerization of actin enzyme-substrate bind-ing for two enzymes and growth of living yeast cells30ndash33

a)Electronic mail sandippiitgernetin

All these findings raised the possibility of TMAO actingas a pressure-counteractant34 A particularly strong stabi-lization of the well-characterized monomeric protein staphy-lococcal nuclease by TMAO against pressure was alsoreported in a high-pressure small angle x-ray scatteringstudy36

Because of its protecting ability against pressure33 34

and urea-induced21 23 protein denaturation the interaction ofTMAO with proteins has been widely studied using variousmodel compounds38ndash43 It was suggested that the interactionbetween TMAO and apolar group is favorable but is over-come by the highly unfavorable interaction between TMAOand the peptide backbone leading to exclusion of TMAOmolecules from the protein surface38 Molecular dynamics(MD) simulations by Zou et al39 showed enhancement of wa-ter structure by TMAO in the form of a slight increase in thenumber of hydrogen bonds per water molecule stronger waterhydrogen bonds and long-range spatial ordering of the sol-vent thereby suggesting protein stabilization via water struc-ture enhancement and hence discouraging hydration of amideunit Although some later simulation studies also revealedthe potential importance of water structure enhancement byTMAO in protein structure protection44 45 other studies ques-tioned the usefulness of this structure enhancement in proteinstabilization46 47

0021-96062012137(9)09450211$3000 copy 2012 American Institute of Physics137 094502-1


094502-2 R Sarma and S Paul J Chem Phys 137 094502 (2012)

As discussed above considerable effort has been madeto understand the protein stabilizing effect of TMAO Nev-ertheless the mode of action of TMAO against pressure-denaturation is not clear yet and this is our primary concernin the present study Again as far as the denaturation of pro-tein by high hydrostatic pressure is concerned we note thatwater penetration into the protein interior at high pressurehas been observed previously15ndash19 and is generally consid-ered to be a primary driving force for pressure-induced pro-tein structural transition In a series of studies Kinoshita andhis co-workers argued that water molecules are extremelycrowded at high pressure and penetration into the protein in-terior increases their total entropy in the system favoring thepressure-denatured structure12ndash14 On the other hand becauseprotein interior is largely composed of hydrophobic residuesit can be assumed easily that hydrophobic contacts are dis-solved at high hydrostatic pressures Indeed pressure-inducedseparation of protein nonpolar groups have been reported inthe literature and there are numerous computational studiesof model hydrophobic solutes suggesting that relative to thecontact state stability of solvent-separated configurations ofthese solutes increases at high pressure48ndash54 Considering theimportance of dissolution of hydrophobic solute in pressure-induced structural transition of protein and to reduce the com-plexity of interactions between solventco-solvent and differ-ent groups of proteins in this study we have investigated thesolvation of hydrophobic solutes at different pressures as afunction of TMAO concentration The influence of TMAO onpressure-induced modifications of hydrogen bond propertiesand dynamics of water molecules around different sphericalshells of the hydrophobic solute surface is also studied hereThe goal is to explore the effect of TMAO on hydrophobicinteraction at high pressure and interpret the protecting effectof TMAO against pressure denaturation of proteins

In a previous article55 we showed that as compared tothe weak association of small methane solutes in water therelatively larger neopentane molecules have much higher as-sociation tendency in water at 1 atm The water hydrogenbonding network was shown to be affected by this larger so-lute and furthermore the effects of pressure on relative dis-persion of hydrophobic solute were found to be amplifiedsignificantly for the larger solute Herein we have presentedresults from MD simulations of neopentane in aqueous solu-tions containing TMAO as well as in pure water over a widerange of pressures First of all we have examined the influ-ence of increasing TMAO contents on the pressure-induceddissolution of hydrophobic solutes in aqueous solution Forthis purpose potentials of mean force (PMF) of neopentanein aqueous solutions are calculated which is complementedby an analysis of association constant To get deeper insightof mode of action of TMAO interaction of neopentane withthe solution species is investigated by computing coordina-tion number and preferential binding parameter The solva-tion characteristics of TMAO molecule at high pressure isalso examined to find the origin of the counteracting effectof TMAO On the other hand effect of TMAO on the waterstructure at high pressure is explored by site-site radial distri-bution functions (rdf) and coordination number calculationsfollowed by an analysis of waterndashwater hydrogen bond prop-

erties and dynamics around different layers of the neopentanemolecule

The remainder of this article is organized into three partsThe models and simulation details are briefly described inSec II the results are discussed in Sec III and our conclu-sions are summarized in Sec IV

II MODELS AND METHODS

Classical MD simulation technique was used to probethe action of TMAO on pressure-induced modification of hy-drophobic interaction and to understand the mechanism ofprotein protection by the osmolyte TMAO against pressuredenaturation The concentration of TMAO in the systems sim-ulated varied from 0 to 4 M and for each TMAO solution sim-ulations were carried out at five different hydrostatic pressuresranging from 1 atm to 8000 atm Initially the 0 M TMAOsolution denoted as system Snp0 was constructed by insert-ing 10 neopentane molecules in a simulation cell containing490 water molecules The water molecules were then replacedwith TMAO (keeping total number of molecules fixed) tocreate three independent TMAO solutions with TMAO molefractions of 003 006 and 008 (denoted as systems Snp2Snp3 and Snp4 respectively) Again to examine waterndashwaterhydrogen bond properties and dynamics 9 of the hydropho-bic solutes were replaced with water molecules and hencethe simulation cell contained only one neopentane moleculein these systems

In all the simulations the extended simple point-charge(SPCE) model56 was used for water the five-site model57 wasadopted for neopentane and the rigid version model of TMAOproposed by Kast et al58 was used In Table I we have sum-marized the values of the Lennard-Jones (LJ) parameters andthe partial charges for neopentane water and TMAO The in-teraction between atomic sites of two different molecules wasexpressed as

uαβ(rαβ) = 4εαβ

[(σαβ

rαβ

)12

minus(

σαβ

rαβ

)6]+ qαqβ

rαβ

(1)

where rαβ is the distance between atomic sites α and β andqα is the charge of the site α The LJ parameters σαβ andεαβ were obtained by using the combining rules σαβ = (σα

+ σβ)2 and εαβ = radicεαεβ

TABLE I The Lennard-Jones parameters and charges used in the modelsconsidered e is the elementary charge

Atom type σ (Aring) ε (kJmol) Charge (e)

Neopentane C 380 0209 00Me 396 06061 00

Water Ow 3166 0646 minus 08476Hw +04238

TMAO Ct 3041 0281 minus 026Nt 2926 08314 +044Ot 3266 06344 minus 065Ht 1775 00769 +011



To obtain the box volume corresponding to the desiredpressure all MD simulations were performed at 298 K initiallyin the isothermal-isobaric (NPT) ensemble for 3 ns Duringthe NPT simulation run the volume of the simulation box wasallowed to fluctuate and the average volume was determinedat the end of the simulation Then the simulations were car-ried out in the NVT ensemble using the box volume obtainedfrom the previous NPT simulation run Each system was equi-librated in NVT ensemble for 5 ns simulation run with velocityrescaling to fix the temperature and finally production sim-ulations for 10 ns were performed Periodic boundary con-ditions and the minimum image convention were used TheLJ interactions were spherically truncated at half-box lengthand the long-range electrostatic interactions were treated us-ing the Ewald method59 The quaternion formulation of theequations of rotational motion was employed and the leap-frog algorithm with a time step of 10minus15 s was used for thetime integration

III RESULTS AND DISCUSSION

A Neopentane-neopentane potentials of mean force

To understand the aggregation of neopentane in aqueoussolutions of increasing TMAO concentration at high pres-sures we have calculated the neopentane-neopentane PMFThe PMF is calculated by using the relation

W (r) = minuskBT ln gss(r) (2)

where gss(r) is the neopentane-neopentane rdf and kB is theBoltzmann constant

The PMF profiles of neopentane are shown inFigure 1 While the first minimum (at about 56 Aring) in thePMF curves arises from the direct contact of two neopen-tane molecules the second minimum reveals the existence of

2 3 4 5 6 7 8 9 10 11 12r (Aring)

-8

-6

-4

-2

0

2

4

6

8

10

12

14

PM

F (

kJm

ol)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa

FIG 1 Potentials of mean force of neopentane as a function of pressure forsystems Snp0 (black) Snp2 (red) Snp3 (green) and Snp4 (blue) For claritythe curves are vertically translated

its solvent-separated state These two minima are thereforereferred to as contact minimum (CM) and solvent-separatedminimum (SSM) respectively We note that in the context ofpressure-induced protein denaturation the contact state cor-responds loosely to the folded state of protein whereas thesolvent-separated state of hydrophobic solutes represents thepressure-denatured state

Figure 1 shows that the well depth of CM is higher thanthat of SSM and the PMF difference between SSM and CM(W ) which is related to the thermodynamic stability of theassociated state or in other words to the association tendencyof hydrophobic solutes in aqueous solution is positive Forexample for system Snp0 at 1 atm pressure the PMF valuesat CM and SSM are minus543 and minus058 kJ molminus1 respectivelygiving a value of +485 kJ molminus1 to W The positive valueof W suggests that a neopentane molecule prefers to be indirect contact with other neopentane moieties rather than tobe separated by a solvent molecule The higher associationtendency of neopentane is also reflected in the positive gradi-ent at larger inter particle distances In regard to the influenceof pressure on the PMF profiles four points are worth noting(a) inward movement of CM SSM and BARR (the maxi-mum between CM and SSM) (b) reduction in the well depthof CM (c) more pronounced free energy basin of solvent-separated state at high pressure and (d) the decrease of theslope of PMF curve at larger solute separation with increas-ing pressure value The W data shown in Figure 2 suggestthat for neopentane in pure water the relative stability of SSMover CM increases at high pressure After an initial sharp de-crease W changes slightly in between 2000 and 6000 atm(at a rate of minus14 mL molminus1) and then its value decreasesdrastically achieving a value of +194 kJ molminus1 at 8000 atm

Now focusing on the influence of TMAO on the PMFof neopentane we find (from Figure 1) that the positions ofCM and BARR do not change in presence of TMAO butSSM shifts to a larger distance Moreover the CM and BARRin the PMF curves shift to upward direction We also ob-serve that TMAO has negligible influence on the magnitudeof the gradient at larger solute separation These results andthe W data shown in Figure 2 do not provide any ev-idence for counteracting effect of TMAO against pressure-denaturation of protein by enhancing hydrophobic associa-

1

2

3

4

5

6

ΔW (

kJ m

ol-1

)

01 200 400 600 800P (MPa)

FIG 2 Thermodynamic stability of associated state of neopentane over itssolvent-separated state as a function of pressure for aqueous solutions of dif-ferent TMAO concentrations Color codes are as in Figure 1 The estimatedstandard deviations for all points fall within plusmn005 to plusmn015 kJmol



tion at high pressure Note that by carrying out a separateMD simulation we also observed a favorable free energy oftransfer (by about 25 kcalmol) of a neopentane moleculefrom pure water to 4 M aqueous TMAO solution at a higherpressure value (6000 atm) which provides corroborative ev-idence of dissolution of neopetane in presence of TMAOMoreover in a previous constraint MD simulation study onthe counteraction of urea by TMAO also it was concludedthat TMAO does not counteract urea denaturation by enhanc-ing hydrophobic interaction60 However as compared to oursimulations relatively higher destabilization of the contact-configuration of neopentane in aqueous TMAO solution wasobserved in that study This discrepancy may be due to theuse of ten neopentane molecules in this study (against two inthat study) to describe the aggregation phenomena which isexpected to be governed by strong dewetting transitions Inthis regard it has been shown that hydrophobic interactionsbetween more than two particles are not pairwise61 62 It wasreported that the solvent contribution to the hydrophobic as-sociation free energy is related to the molecular surface ofthe hydrophobic cluster62 In a later study Lum et al63 alsoreported that for large local concentration of solute parti-cles or when each solute particle has a sufficiently large sur-face which can induce drying leading to strong attractionsbetween the nonpolar solutes Our observations that TMAOdoes not have any substantial effect on hydrophobic interac-tions as revealed by the PMF plots is on the other hand sup-ported by the simulation study of Athawale et al46

For a better understanding on the effect of increasingTMAO concentration on neopentane aggregation at high pres-sures we have computed the association constant Ka whichis defined as

Ka = 4π

int ra

0r2eminusW (r)kBT dr (3)

where ra is the position of the BARR in the correspondingPMF curve Note that a higher value of Ka indicates greaterassociation tendency of hydrophobic moieties in aqueous sys-tem Any perturbant that favors the solvent-separated state ofhydrophobic solute will reduce the value of Ka

The values of Ka for different systems investigated hereare presented in Table II From this table we can see that themagnitude of Ka reduces with increasing pressure Again ata particular pressure the values of Ka are lower in presenceof TMAO (as compared to pure water system) These resultsclearly indicate that neopentane molecules are relatively dis-

TABLE II Association constant of neopentane (in the unit of Mminus1) for thesystems investigated Numbers in system names indicate approximate TMAOconcentration (in M)

System

P (MPa) Snp0 Snp2 Snp3 Snp4

01 443 441 438 420200 220 190 188 183400 209 167 167 153600 171 168 122 113800 111 123 107 091

persed at high pressure and TMAO does not act as a stabiliz-ing co-solvent for hydrophobic interaction at high pressure

B Site-site radial distribution function

To obtain molecular details of neopentane solvation wehave computed site-site rdfs between neopentane and solu-tion species average number of solventco-solvent moleculesaround the hydrocarbon solute and then the preferentialbinding parameter The number of solution species aroundneopentane is calculated by using the relation

nαβ = 4πρβ

int r2

r1

r2gαβ(r)dr (4)

where nαβ represents the number of atoms of type β surround-ing atom α in a shell extending from r1 to r2 and ρβ is thenumber density of β in the system For the calculation of firstshell coordination number r1 and r2 are zero and the locationof the first minimum in the corresponding rdf respectively

Figure 3 displays rdfs that concern solvation of neopen-tane The effects of pressure on neopentane hydration in purewater system were discussed recently in detail in Ref 55 Inshort the first peak and the first minimum in the neopentanendashwater rdf shift to shorter distances and the height of the firstpeak increases monotonically with pressure indicating effi-cient packing of water molecules around the solute at highpressure The hydration number of neopentane (Table III)also reveals that at high pressure number of water moleculesaround neopentane increases These results are in agreementwith the general findings of nonpolar group separation by wa-ter molecules at high pressure48ndash53

0123456789

101112

g(r)

3 4 5 6 7 8 9 10 11r (Aring)

0123456789

101112

g(r)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa

FIG 3 Neopentane-water oxygen (top) and neopentane-TMAO nitrogen(bottom) rdfs as a function of pressure for systems Snp0 (black) Snp2 (red)and Snp4 (blue)



TABLE III Average number of water (nw) and TMAO (nt) molecules inthe first coordination shell of neopentane (obtained by integrating the rdf tothe first minimum) and the preferential binding parameter νwt

System P (MPa) nw nt νwt

Snp0 01 202 Snp0 200 261 Snp0 400 261 Snp0 600 270 Snp0 800 288

Snp2 01 180 (196) 16 minus06Snp2 200 241 (253) 16 minus05Snp2 400 244 (253) 18 minus06Snp2 600 242 (262) 18 minus06Snp2 800 253 (279) 18 minus06



The height and location of the first peak remain almostunaffected on addition of TMAO Nonetheless the coordi-nation numbers suggest TMAO-induced removal of watermolecules from the solvation shell of neopentane (Table III)Since some reduction in the hydration number of neopentanein aqueous TMAO solutions arises from the reduced num-ber density of water molecules in the system we have alsocalculated the coordination numbers assuming that the onlychange with added TMAO comes through the reduced num-ber of water and these numbers are also given in Table IIIin parentheses The replacement of water by TMAO is clearfrom these values Moreover we find that the ratio of the hy-dration number of neopentane in pure water and in aqueoussolution of TMAO is higher than the respective ratio of watermolecules present in those systems For example the ratio offirst-shell water molecules in systems Snp0 and Snp4 at 1 atmis 131 1 which is higher than the ratio of total number of wa-ter molecules present in those systems (109 1) This againreveals TMAO-induced removal of water molecules from thesolvation shell of neopentane

Focusing on the interaction of TMAO with neopentane(Figure 3) we find that as compared to that in neopentanendashwater rdf the first peak is stronger in neopentanendashTMAOrdf This observation suggests that neopentane prefers to in-teract more with TMAO than with water We also observethat the first peak in neopentanendashTMAO rdf reduces at higherTMAO concentration In Table III we have presented theaverage number of TMAO molecules in the first solvationshell of neopentane It can be seen easily that the numberof TMAO molecules in the vicinity of neopentane increaseswith increasing TMAO concentration The number also in-creases at high pressure but the change is relatively smallThis enhancement of TMAO molecules (due to increasing

TMAO concentration) around neopentane is likely why wa-ter molecules are excluded from the neopentane surface

To examine the solvation of neopentane more closely wehave computed the preferential binding parameter νwt usingthe relation64

νwt = nwNw

ntNt

minus 1 (5)

where n is the number of molecules in the vicinity of neopen-tane while N represents the total number of molecules inthe system with subscripts w and t standing for water andTMAO respectively Note that the number of molecules inthe vicinity of neopentane ni is obtained by integrating thecorresponding rdf to the first minimum which gives the outerlimit of the first-solvation shell This binding parameter char-acterizes the extent of accumulation or exclusion of a solutionspecies from the vicinity of the hydrophobic solute surfacerelative to the bulk While a positive value of ναβ indicatespreference of the solution constituent for the species α overthe species β the value becomes negative in case of preferen-tial exclusion of the species α Results obtained for neopen-tane are included in Table III The negative value of νwt con-firms more preference of neopentane for TMAO over waterWe further note that this parameter is not a strong function ofeither pressure or TMAO concentration which suggests thatthe preferential binding of neopentane with TMAO moleculesdoes not change with pressure or TMAO concentration

In a previous article65 we examined the orientation ofTMAO molecules near the neopentane moiety by computingall the rdfs involving neopentane and TMAO at 1 atm and itwas found that TMAO molecules prefer a side-on orientationnear the neopentane surface allowing its oxygen atom to formfavorable hydrogen bonds with water as well as maintain-ing some hydrophobic contacts with the hydrophobic moietyHere in Figure 4 we have included selected site-site rdfs thatshow the influence of pressure on the orientation of TMAOaround the surface of neopentane From Figure 4 we see thatthe nitrogen (Nt ) and carbon (Ct ) atoms of TMAO show onlyslight awareness to high pressure What influence there isis mainly apparent in the orientation of TMAO oxygen (Ot )Specifically the first peak of oxygen density profile which isresponsible for hydrogen bonding with water molecules nearneopentane surface becomes more pronounced at high pres-sure and the location of it shifts toward shorter distance Thischange in the orientation of oxygen atom can be related di-rectly to the enhanced hydration of neopentane at high pres-sure On the other hand the significantly small TMAO den-sity (weak peak in these rdfs) around neopentane at 1 atmis correlated with the large aggregation of neopentane at thispressure

To obtain information regarding the hydration of TMAOas a function of its concentration at different pressures wehave also examined the behavior of water molecules in thevicinity of both hydrophobic (methyl group) and hydrogenbonding (oxygen atom) sites of TMAO Results obtained areshown in Figure 5 Focusing on the hydration of TMAOat 1 atm first we find that the first peaks in Ct minus Ow andCt minus Hw profiles appear at similar locations as observed pre-viously in simulations of TMAO solution at 1 atm46 This



0

05

1

15

2

25

3

g(r)

0

05

1

15

2

25

3

g(r)

2 3 4 5 6 7 8 9 10 11 12r(Aring)

0

05

1

15

2

25

3

g(r)

FIG 4 Site-site interaction of neopentane with nitrogen (Nt ) oxygen (Ot )and carbon (Ct ) atoms of TMAO (from top to bottom respectively) for 4 MTMAO solution (system Snp4) at 1 (black) 2000 (red) 4000 (green) 6000(blue) and 8000 (magenta) atm

behavior is typical of hydrophobic hydration and indicatessurface-parallel orientation of water molecules around the hy-drophobic surface On the other hand the orientational char-acteristics of water molecules (preferred water hydrogen overwater oxygen) near the TMAO oxygen are very similar tothat near a negative ion or near a water oxygen in bulk waterThe strong first peaks in Ot minus Ow and Ot minus Hw rdfs demon-strate strong affinity of TMAO for water coming throughTMAOndashwater hydrogen bonding The height of the first peak(at about 18 Aring ) for Ot minus Hw rdf is much higher than that forOw minus Hw rdf (Figure 6) This immediately implies that wa-ter has higher tendency to be hydrogen bonded to a TMAOmolecule than to a second water molecule We note that sig-nificantly longer hydrogen bond life-time for a TMAOndashwaterthan for a waterndashwater hydrogen bond has been widely re-ported in the literature39 66ndash69 The MD simulations also pre-dicted that TMAOndashwater hydrogen bonds are more attractivethan waterndashwater hydrogen bonds69

Integration of the rdfs gives 71 and 27 water moleculesin the first hydration shell of TMAO methyl group and oxygenatom respectively for the system Snp4 at 1 atm These num-bers are in broad concordance with previous studies66 70 71

Addition of these numbers results in a molecular hydrationnumber of 240 (total number of water molecules around threemethyl groups plus one oxygen atom of TMAO) Consider-ing the fact that some of these water molecules are sharedbetween different groups the hydration number per TMAOmolecule agrees well with Meersman et al71 On the otherhand in an infrared spectroscopy study it was observed thatabout 85 water molecules are affected by TMAO72 Sincesuch a discrepancy in hydration number can be a result of

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

30

60

90

120

150

180

runn

ing

coor

d n

o

0

30

60

90

120

150

180

runn

ing

coor

d n

o

FIG 5 TMAO carbon-water oxygen (left) and TMAO oxygen-water hydro-gen (right) site-site rdfs as a function of pressure for systems Snp2 (top) andSnp4 (bottom) The running coordination numbers of water oxygen aroundcarbon (left) and oxygen (right) atoms of TMAO are also shown for thesetwo systems Different colors are as in Figure 4 Orange colored lines arefor TMAO carbon-water hydrogen (left) and TMAO oxygen-water oxygen(right) rdf at 1 atm

0

1

2

3

4

g(r)

0

1

2

3

4

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

1

2

3

4

g(r)

FIG 6 Water oxygen-water hydrogen site-site rdfs as a function of pressurefor systems Snp0 Snp2 and Snp4 (from top to bottom respectively) Differ-ent colors are as in Figure 4 Orange colored lines are for water oxygen-wateroxygen rdf at 1 atm



different definitions we have also calculated the hydrationnumber by integrating the corresponding rdf only to the firstmaximum (instead of using the first hydration shell) Thenumbers obtained by this method for the system Snp4 at 1 atmare 22 and 09 for TMAO methyl group and oxygen atom re-spectively yielding an average hydration number of 75 perTMAO molecule This is in line with the experimental obser-vation

The height of the first peak of Ct minus Ow rdf increasesslightly with increasing TMAO concentration At high pres-sure the first peak and minimum shift to shorter distances andwhile the height of the first peak increases monotonically withpressure the first minimum shows a downward movementThese observations are very similar to the effect of pressureon hydration of neopentane and reflect enhanced packing ofwater molecules around methyl groups of TMAO at highpressure

The number density of water molecules also increases inthe vicinity of TMAO oxygen with increasing concentrationof TMAO Nevertheless unlike the hydrophobic hydrationthe TMAOndashwater hydrogen bonding interaction remains al-most unaffected by pressure The location and height of thefirst peak do not show any change and the first valley showsa slightly upward (and also inward) movement To get insightinto the nature of hydrogen bonding we have calculated thenumber of hydrogen bonded water molecules to TMAO oxy-gen by integrating the rdf to the first minimum and the num-bers obtained are presented in Table IV The data show thaton average there are about three water molecules that donatehydrogen atoms to TMAO oxygen which is consistent with

TABLE IV Coordination numbers for atom pairs associated with hydrogenbonding W and T refer to water and TMAO respectively Numbers are withrespect to the first species and the total is calculated by taking into accountthe number of hydrogen atoms in water

PW-W W-T T-W

System (MPa) OndashH HndashO Total OndashH HndashO Total OndashH HndashO Total

Snp0 01 099 099 396 Snp0 200 099 099 396 Snp0 400 099 099 396 Snp0 600 099 099 396 Snp0 800 099 099 396

Snp2 01 092 092 368 004 008 138 276Snp2 200 098 098 392 004 008 140 280Snp2 400 098 098 392 004 008 142 284Snp2 600 098 098 392 005 010 143 286Snp2 800 098 098 392 005 010 143 286



the notion that TMAO oxygen is hydrated by two to three wa-ter molecules42 46 66 71 73ndash75

Negligible enhancement in the number of hydrogenbonded water molecules is observed at high pressure On theother hand although the number reduces per TMAO oxy-gen with increasing TMAO concentration the total numberof TMAOndashwater hydrogen bonds increases For example thetotal number of Ow minus Hw middot middot middot Ot hydrogen bonds for systemSnp2 at 1 and 8000 atm are 4140 (276 times 15) and 4290 (286times 15) respectively whereas the same for system Snp4 at 1and 8000 atm are 10400 (260 times 40) and 11040 (276 times 40)respectively The examination of solvation characteristics ofTMAO thus suggests that TMAO is well solvated by watermolecules and the number of water molecules required to sol-vate all TMAO molecules present in the system increases withincreasing TMAO contents

An examination of possible effects of pressure andTMAO on hydrogen bonding network of water (Figure 6)shows that the Ow minus Hw profiles are affected only slightlyby pressure Integration of the rdf to the first minimum alsosuggests negligible effect of pressure on the number of hy-drogen bonded water molecules (Table IV) TMAO in con-trast enhances the first peak Coordination numbers listed inTable IV shows that for a particular pressure waterndashwater hy-drogen bonds reduces with increasing TMAO concentrationand these hydrogen bonds are replaced by TMAOndashwater hy-drogen bonds While TMAO-induced replacement of waterndashwater hydrogen bonds has been discussed extensively in theliterature42 69 a somewhat opposite result was reported byZou et al39

C Hydrogen bond properties and dynamics

From the solution structure analysis discussed aboveit appears that on average each water molecule can formabout four hydrogen bonds with identical species Pressuredoes not affect the number of waterndashwater hydrogen bondswhereas TMAO reduces these hydrogen bonds by forming it-self hydrogen bonds with water Further insight into the na-ture of waterndashwater hydrogen bonding interactions in aque-ous solutions of TMAO as a function of TMAO concentrationand pressure can be obtained by investigating the propertiesand dynamics of these hydrogen bonds in different layersaround the hydrophobic solute In an attempt to this we con-sidered two regions from the vicinity of the neopentane sur-face (Region I 30ndash40 Aring and Region II 40ndash50 Aring) anda third region was considered at a relatively larger distancefrom the solute assuming bulk portion of the simulation cell(Region III 100ndash110 Aring) The methods for hydrogen bondproperties and dynamics calculations were identical to thosedescribed in Ref 65 A water pair was considered to be hy-drogen bonded if Ow minus Ow and Ow minus Hw distances are lessthan 34 and 24 Aring respectively and simultaneously theOw minus Ow minus Hw angle is less than 45

In Table V we have presented the average number ofwaterndashwater hydrogen bonds per water molecule togetherwith hydrogen bond life-time and energy of these bonds Wenote that at 1 atm pressure in pure water the number of



TABLE V Waterndashwater hydrogen bond properties and dynamics nHB EHB and τHB represent average numberenergy (in kJ molminus1) and life-time (in ps) respectively Regions I II and III are defined in the text

System P (MPa) nIHB nII

HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB

Snp0 01 337 363 378 minus2013 minus1951 minus1873 155 146 150Snp0 200 356 376 388 minus2005 minus1941 minus1828 145 138 135Snp0 400 367 387 396 minus1964 minus1892 minus1802 125 129 137Snp0 600 372 394 402 minus1941 minus1885 minus1782 123 122 129Snp0 800 382 398 406 minus1926 minus1858 minus1753 121 116 126




hydrogen bonded water molecules in the bulk region (Re-gion III) is 378 which is in concordance with that obtained(about 345) by Huang et al76 taking into account the use ofdifferent water models (SPCE vs SPC) and hydrogen bondcriteria (45 vs 30) Three points visible from Table Vare (a) in order to accommodate large neopentane solutesome of the waterndashwater hydrogen bonds must be brokenand as a result of this number of waterndashwater hydrogenbonds reduces in fraction in the vicinity of the solute ascompared to the bulk region at the same time near the so-lute surface these bonds are more attractive (b) TMAO re-duces the average number of waterndashwater hydrogen bondsand makes hydrogen bonds slightly stronger and (c) pressureinduced enhancement of average number of waterndashwater hy-drogen bonds and reduction of waterndashwater hydrogen bondenergy Pressure-induced reduction and TMAO-induced en-hancement in waterndashwater hydrogen bond life-time can alsobe seen from Table V This modification of life-expectancy ofwaterndashwater hydrogen bond is further reflected in the decay ofcontinuous hydrogen bond time correlation function SHB(t)with time as shown in Figure 7 This figure shows that the hy-drogen bond decays slightly faster at high pressure and the de-cay is much slower for highly concentrated TMAO solutionTMAO-induced enhancement of waterndashwater hydrogen-bondlife-time was reported in previous MD simulations39 69 77

Based on MD simulations on TMAOndashwater clusters Weiet al45 also suggested that TMAO strengthens the hydrogenbonds between water molecules and slows down the orien-tational relaxation of water significantly They predicted thatthe methyl groups on TMAO are responsible for these effectsHere we also note that Rezus and Bakker78 79 in their infra-red pump probe experiment studies of water-TMAO mixtureshowed that the orientational mobility of water moleculesnear to TMAO methyl group is greatly reduced compared to

bulk water molecules Another experimental study also sug-gested that the rotational dynamics of water molecules areslowed down in presence of TMAO and as the concentrationincreases the dynamics are further decelerated66

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)

FIG 7 Time-dependence of continuous waterndashwater hydrogen bond corre-lation function SHB(t) as a function of pressure around different sphericalshells of neopentane for systems Snp0 Snp2 and Snp4 (from top to bottomrespectively) Different colors are as in Figure 4 Regions I II and III aredefined in the text



001020304050607

f5

Region I Region II Region III

001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800

P (MPa) P(MPa) P(MPa)

FIG 8 The fraction of water with n number of hydrogen bonds as a functionof pressure around different spherical shells of neopentane for systems Snp0(black) Snp2 (red) and Snp4 (blue) Regions I II and III are defined in thetext

In Figure 8 we have shown the variation of fraction ofwater molecules (fn with n number of hydrogen bonds) as afunction of TMAO concentration as well as of pressure forthe three selected regions At a particular pressure the pertur-bation in hydrogen bonding network of water molecules cre-ated by the relatively large neopentane molecule can be seenfrom the enhancement in the fraction of 2 and 3 coordinatedwater molecules while reducing the fraction of 4 and 5 coordi-nated water molecules in the vicinity of this hydrophobic so-lute (Regions I and II) as compared to the bulk region (RegionIII) This clearly suggests that water loses some of its identicalnearest neighbors to accommodate the hydrophobic soluteEnhancement of pressure increases the fraction of higher co-ordinated (4 and 5) water molecules and reduces the fractionof lower coordinated (2 and 3) water molecules indicatingwater crowding at high pressure We note that the pressure-induced water crowding at high pressure was suggested to bea possible reason for water penetration (which relaxes watercrowding in the bulk) into the protein interior causing proteindenaturation12ndash14 We also find TMAO-induced reduction inthe number of identical nearest neighbors of water moleculesThe loss in the number of higher coordinated water moleculesis correlated with the solvation of large TMAO moleculesThe significant enhancement in the number of 2 and 3 coordi-nated water molecules and reduction in the number of 4 and 5

coordinated water molecules for the highest TMAO concen-tration investigated here on the other hand provides corrob-orative evidence for our observation (from coordination num-ber) that the number of water molecules that solvate TMAOincreases with increasing concentration of TMAO

IV SUMMARY AND CONCLUSIONS

In this article hydrophobic effect is investigated in aque-ous TMAO solutions at different pressures to understand themechanism of protein protection by TMAO at high pressureUsing neopentane as model of hydrophobic solute molecu-lar dynamics simulations are carried out for four indepen-dent TMAO solutions each at five different pressures rele-vant to pressure induced protein denaturation Potentials ofmean force and the relative stability of the associated stateof hydrophobic solute over its solvent-separated state are cal-culated It is found that neopentane molecules prefer self-association at low pressure and high pressure causes rela-tive dispersion of these hydrophobic moieties Supportingthe notion that nonpolar side-chains are separated by watermolecules at high pressure48ndash53 our simulation results alsoshow that pressure increases compactness of neopentane hy-dration shell and as compared to the low pressure system athigh pressure water molecules are preferred in the solvationshell of neopentane On the other hand relative to pure waterthe hydrophobic association of neopentane does not improvein presence of TMAO

From investigations of waterndashwater hydrogen bond prop-erties and dynamics in different layers around the solutemolecule it is observed that due to the large size of neopen-tane water molecule loses a fraction of identical neigh-bors in the vicinity of the hydrophobic solute as comparedto the bulk region At high pressure water molecules areforced to occupy interstitial positions leading to an increasein the number of weakly hydrogen bonded water moleculesThe number of five-coordinated water molecules now in-creases and that of three-coordinated decreases In our pre-vious study55 we predicted that to reduce the restrictionin movement arising from this crowding water moleculesshow a tendency to occupy the space between neopentanemolecules and the solute separation gives an entropic profitto the system Extending to pressure-denaturation of pro-teins it has been suggested that the denaturation processis largely contributed by the relaxation of water moleculesfar away from the protein surface resulting from the wa-ter penetration into the tightly packed hydrophobic pro-tein interior12ndash14 In aqueous TMAO solution the numberof identical nearest neighbor water molecules decreasesThis is reflected in our waterndashwater hydrogen bond analy-sis showing that the average number of higher coordinatedwater molecules decreases in presence of TMAO Further-more in accordance with an earlier study69 our coordinationnumber calculations also show that there is formationof considerable number of water-TMAO hydrogen bonds(more than two hydrogen bonds per TMAO molecules)Additionally as it is evident from stronger waterndashwater hy-drogen bond energies and enhancement of waterndashwater hy-drogen bond life times the hydrogen bonding network of



water becomes stronger in presence of TMAO So thedirect interaction of TMAO with water and its effect on waterhydrogen bonding network greatly reduces the need of watermolecules to penetrate into the hydrophobic interior Hence itis reasonable to expect that TMAO can reduce the pressure-induced enhancement of hydrophobic hydration Indeed it isreflected in our coordination number analysis showing thatneopentane is dehydrated in aqueous solution of TMAO Butwhat seems to be unexpected is the observation that TMAOdoes not reduce the pressure-induced dispersion of neopen-tane molecules Instead TMAO is likely to reduce neopentaneaggregation which is a clear consequence of direct interactionof TMAO with the neopentane moieties

Finally combining our simulation results with the sug-gestion that the highly unfavorable interaction of TMAOwith the peptide backbone can easily overcome its favorableinteraction with protein side-chains leading to TMAO exclu-sion from the protein surface38 we could explain the TMAO-induced counteraction of protein denaturation at high hydro-static pressures as follows Because TMAO molecules aremostly excluded from the protein surface these are free to besolvated by water molecules Indeed each TMAO moleculeforms hydrogen bonds with two to three water moleculesreducing their availability to solvate the protein sites Moreimportantly TMAO molecules do not show self-aggregationin water and TMAO-water hydrogen bonds are reported tobe stronger than waterndashwater hydrogen bonds69 70 We expectthese exceptional properties of TMAO to contribute signif-icantly to its counteracting ability In addition to the directTMAOndashwater interaction TMAO also shows an indirect ef-fect in which it makes the hydrogen bonding network of wa-ter stronger leading to reduction of water penetration into theprotein interior We would like to conclude that the direct in-teraction of TMAO with water and its (TMAO) indirect effecton the water structure are the most likely reasons for counter-action of pressure-induced protein denaturation In our opin-ion TMAO does not need any direct interaction with proteinfor its counteracting effect We hope that modeling of morerealistic protein systems will be useful in shedding new lightin this direction

ACKNOWLEDGMENTS

The financial support of the Department of Science andTechnology (DST) Government of India is gratefully ac-knowledged The author RS thanks the Council of Scien-tific and Industrial Research (CSIR) India for providing fel-lowship

1J Zhang X Peng A Jonas and J Jonas Biochemistry 34 8631 (1995)2G J A Vidugiris J L Markely and C A Royer Biochemistry 34 4909(1995)

3K Akasaka H Li H Yamada R Li T Thoresen and C K WoodwardProtein Sci 8 1946 (1999)

4M Marchi and K Akasaka J Phys Chem B 105 711 (2001)5S D Samarasinghe D M Campbell A Jonas and J Jonas Biochemistry31 7773 (1992)

6K Akasaka T Tezuka and H Yamada J Mol Biol 271 671 (1997)7H Li H Yamada and K Akasaka Biochemistry 37 1167 (1998)8H Li H Yamada and K Akasaka Biophys J 77 2801 (1999)9G Panick R Melissa R Winter G Rapp K J Frye and C A Royer JMol Biol 275 389 (1998)

10W B Floriano M A C Nascimento G B Domont and W A GoddardIII Protein Sci 7 2301 (1998)

11N Smolin and R Winter J Phys Chem B 112 997 (2008)12Y Harano and M Kinoshita J Chem Phys 125 024910 (2006)13Y Harano T Yoshidome and M Kinoshita J Chem Phys 129 145103

(2008)14T Yoshidome Y Harano and M Kinoshita Phys Rev E 79 011912

(2009)15T Imai S Ohyama A Kovalenko and F Hirata Protein Sci 16 1927

(2007)16J R Grigera and A N McCarthy Biophys J 98 1626 (2010)17M W Lasalle H Yamada and K Akasaka J Mol Biol 298 293 (2000)18R Day and A E Garciacutea Proteins Struct Funct Bioinf 70 1175 (2008)19T Imai and Y Sugita J Phys Chem B 114 2281 (2010)20T-Y Lin and S N Timasheff Biochemistry 33 12695 (1994)21V Daggett Chem Rev 106 1898 (2006)22L M Samuelsson J J Bedford R A J Smith and J P Leader Comp

Biochem Physiol A 141 22 (2005)23P Venkatesu M-J Lee and H-m Lin J Phys Chem B 113 5327 (2009)24R D Lins C S Pereira and P H Huumlnenberger Proteins Struct Funct

Bioinf 55 177 (2004)25N K Jain and I Roy Protein Sci 18 24 (2009)26H Herberhold C A Royer and R Winter Biochemistry 43 3336 (2004)27M B Gillett J R Suko F O Santoso and P H Yancey J Exp Zool

279 386 (1997)28R H Kelly and P H Yancey Biol Bull 196 18 (1999)29J R Treberg and W R Driedzic J Exp Zool 293 39 (2002)30P H Yancey M D Rhea K M Kemp and D M Bailey Cell Mol Biol

50 371 (2004)31P H Yancey and J F Siebenaller J Exp Biol 202 3597 (1999)32P H Yancey W Blake and J Conley Comp Biochem Physiol A 133

667 (2002)33P H Yancey A L Fyfe-Johnson R H Kelly V P Walker and M T

Auntildeoacuten J Exp Zool 289 172 (2001)34P H Yancey J Exp Biol 208 2819 (2005)35P H Yancey Am Zool 41 699 (2001)36C Krywka C Sternemann M Paulus M Tolan C Royer and R Winter

Chem Phys Chem 9 2809 (2008)37T Yamazaki T Imai F Hirata and A Kovalenko J Phys Chem B 111

1206 (2007)38A Wang and D W Bolen Biochemistry 36 9101 (1997)39Q Zou B J Bennion V Daggett and K P Murphy J Am Chem Soc

124 1192 (2002)40S L Lin A Z Afsar and A R Davidson Protein Sci 18 526 (2009)41C Y Hu G C Lynch H Kokubo and B M Pettitt Proteins 78 695

(2010)42H Kokubo C Y Hu and B M Pettitt J Am Chem Soc 133 1849

(2011)43S S Cho G Reddy J E Straub and D Thirumalai J Phys Chem B

115 13401 (2011)44L Yang and Y Q Gao J Am Chem Soc 132 842 (2010)45H Wei Y Fan and Y Q Gao J Phys Chem B 114 557 (2010)46M V Athawale J S Dordick and S Garde Biophys J 89 858 (2005)47M V Athawale S Sarupria and S Garde J Phys Chem B 112 5661

(2008)48G Hummer S Garde A E Garciacutea M E Paulaitis and L R Pratt Proc

Natl Acad Sci USA 95 1552 (1998)49A Wallqvist J Chem Phys 96 1655 (1992)50A Wallqvist Chem Phys Lett 182 237 (1991)51A Wallqvist J Phys Chem 95 8921 (1991)52V A Payne N Matubayasi L R Murphy and R M Levy J Phys Chem

B 101 2054 (1997)53T Ghosh A E Garciacutea and S Garde J Am Chem Soc 123 10997

(2001)54S W Rick J Phys Chem B 104 6884 (2000)55R Sarma and S Paul J Chem Phys 136 114510 (2012)56H J C Berendsen J R Grigera and T P Straatsma J Phys Chem 91

6269 (1987)57W L Jorgensen J D Madura and C J Swenson J Am Chem Soc 106

6638 (1984)58K M Kast J Brickmann S M Kast and R S Berry J Phys Chem A

107 5342 (2003)59M P Allen and D J Tildesley Computer Simulation of Liquids (Claren-

don Oxford 1987)



60S Paul and G N Patey J Phys Chem B 112 11106 (2008)61G Neacutemethy and H A Scheraga J Phys Chem 66 1773 (1962)62J A Rank and D Baker Protein Sci 6 347 (1997)63K Lum D Chandler and J D Weeks J Phys Chem B 103 4570

(1999)64R Zangi R Zhou and B J Berne J Am Chem Soc 131 1535

(2009)65R Sarma and S Paul J Chem Phys 135 174501 (2011)66J Hunger K-J Tielrooij R Buchner M Bonn and H J Bakker J Phys

Chem B 116 4783 (2012)67H-S Lee and M E Tuckerman J Chem Phys 126 164501 (2007)68G Stirnemann J T Hynes and D Laage J Phys Chem B 114 3052

(2010)69S Paul and G N Patey J Am Chem Soc 129 4476 (2007)70S Paul and G N Patey J Phys Chem B 110 10514 (2006)

71F Meersman D Bowron A K Soper and M H J Koch Phys ChemChem Phys 13 13765 (2011)

72A Panuszko P Bruzdziak J Zielkiewicz D Wyrzykowski and J Stan-gret J Phys Chem B 113 14797 (2009)

73J Roumlsgen and R Jackson-Atogi J Am Chem Soc 134 3590 (2012)74F Meersman D Bowron A K Soper and M H J Koch Biophys J 97

2559 (2009)75K L Munroe D H Magers and N I Hammer J Phys Chem B 115

7699 (2011)76X Huang C J Margulis and B J Berne J Phys Chem B 107 11742

(2003)77B J Bennion and V Daggett Proc Natl Acad Sci USA 101 6433

(2004)78Y L A Rezus and H J Bakker Phys Rev Lett 99 148301 (2007)79Y L A Rezus and H J Bakker J Phys Chem B 113 4083 (2009)


THE JOURNAL OF CHEMICAL PHYSICS 137 094502 (2012)

The effect of aqueous solutions of trimethylamine-N-oxide on pressureinduced modifications of hydrophobic interactions

Rahul Sarma and Sandip Paula)

Department of Chemistry Indian Institute of Technology Guwahati Assam 781039 India

(Received 28 May 2012 accepted 13 August 2012 published online 4 September 2012)

To understand the mechanism of protein protection by the osmolyte trimethylamine-N-oxide(TMAO) at high pressure using molecular dynamics (MD) simulations solvation of hydrophobicgroup is probed in aqueous solutions of TMAO over a wide range of pressures relevant to proteindenaturation The hydrophobic solute considered in this study is neopentane which is a consider-ably large molecule The concentrations of TMAO range from 0 to 4 M and for each TMAO con-centration simulations are performed at five different pressures ranging from 1 atm to 8000 atmPotentials of mean force are calculated and the relative stability of solvent-separated state over theassociated state of hydrophobic solute are estimated Results suggest that high pressure reduces asso-ciation of hydrophobic solutes From computations of site-site radial distribution function followedby analysis of coordination number it is found that water molecules are tightly packed around thenonpolar particle at high pressure and the hydration number increases with increasing pressure Onthe other hand neopentane interacts preferentially with TMAO over water and although hydrationof neopentane reduces in presence of this osmolyte TMAO does not show any tendency to preventthe pressure-induced dispersion of neopentane moieties It is also observed that TMAO moleculesprefer a side-on orientation near the neopentane surface allowing its oxygen atom to form favorablehydrogen bonds with water while maintaining some hydrophobic contacts with neopentane Analy-sis of hydrogen-bond properties and solvation characteristics of TMAO reveals that TMAO can formhydrogen bonds with water and it reduces the identical nearest neighbor water molecules caused byhigh hydrostatic pressures Moreover TMAO enhances life-time of waterndashwater hydrogen bonds andmakes these hydrogen bonds more attractive Implication of these results for counteracting effect ofTMAO against protein denaturation at high pressures are discussed copy 2012 American Institute ofPhysics [httpdxdoiorg10106314748101]

I INTRODUCTION

It is well known that the stability of native proteins inaqueous solution depends strongly on the thermodynamicvariables such as pressure1ndash19 as well as on the surround-ing chemical environments20ndash25 and in recent years effectof chemical agents on protein structure and stability asso-ciated with pressure denaturation has received increasinginterest26ndash37 Different co-solvents have well-documented ef-fects on protein stability at high pressure Chaotropic agentssuch as urea make protein to unfold at a lower pressure ascompared to the pure water system26 In contrast polyhy-dric alcohols (glycerol sorbitol) can increase the denatura-tion pressure26 It has been reported that the concentrationof a naturally occurring osmolyte trimethylamine-N-oxide(TMAO) increases linearly with depth of the sea both amongdifferent species and within the same species while reduc-ing the concentration of the common osmolytes of shallowrelatives eg glycine in shrimp urea in skates27ndash30 Thisparticular osmolyte (TMAO) was found to offset pressure-inhibition of stability of several lactate dehydrogenase ho-mologues polymerization of actin enzyme-substrate bind-ing for two enzymes and growth of living yeast cells30ndash33

a)Electronic mail sandippiitgernetin

All these findings raised the possibility of TMAO actingas a pressure-counteractant34 A particularly strong stabi-lization of the well-characterized monomeric protein staphy-lococcal nuclease by TMAO against pressure was alsoreported in a high-pressure small angle x-ray scatteringstudy36

Because of its protecting ability against pressure33 34

and urea-induced21 23 protein denaturation the interaction ofTMAO with proteins has been widely studied using variousmodel compounds38ndash43 It was suggested that the interactionbetween TMAO and apolar group is favorable but is over-come by the highly unfavorable interaction between TMAOand the peptide backbone leading to exclusion of TMAOmolecules from the protein surface38 Molecular dynamics(MD) simulations by Zou et al39 showed enhancement of wa-ter structure by TMAO in the form of a slight increase in thenumber of hydrogen bonds per water molecule stronger waterhydrogen bonds and long-range spatial ordering of the sol-vent thereby suggesting protein stabilization via water struc-ture enhancement and hence discouraging hydration of amideunit Although some later simulation studies also revealedthe potential importance of water structure enhancement byTMAO in protein structure protection44 45 other studies ques-tioned the usefulness of this structure enhancement in proteinstabilization46 47

0021-96062012137(9)09450211$3000 copy 2012 American Institute of Physics137 094502-1











[(σαβ

rαβ

)12

minus(

σαβ

rαβ

)6]+ qαqβ

rαβ

(1)





Neopentane C 380 0209 00Me 396 06061 00

Water Ow 3166 0646 minus 08476Hw +04238











2 3 4 5 6 7 8 9 10 11 12r (Aring)

-8

-6

-4

-2

0

2

4

6

8

10

12

14

PM

F (

kJm

ol)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa





1

2

3

4

5

6

ΔW (

kJ m

ol-1

)

01 200 400 600 800P (MPa)






Ka = 4π

int ra





System


01 443 441 438 420200 220 190 188 183400 209 167 167 153600 171 168 122 113800 111 123 107 091




nαβ = 4πρβ

int r2

r1

r2gαβ(r)dr (4)



0123456789

101112

g(r)

3 4 5 6 7 8 9 10 11r (Aring)

0123456789

101112

g(r)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa














νwt = nwNw

ntNt

minus 1 (5)






0

05

1

15

2

25

3

g(r)

0

05

1

15

2

25

3

g(r)

2 3 4 5 6 7 8 9 10 11 12r(Aring)

0

05

1

15

2

25

3

g(r)





0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

30

60

90

120

150

180

runn

ing

coor

d n

o

0

30

60

90

120

150

180

runn

ing

coor

d n

o


0

1

2

3

4

g(r)

0

1

2

3

4

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

1

2

3

4

g(r)








PW-W W-T T-W
















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)

























[(σαβ

rαβ

)12

minus(

σαβ

rαβ

)6]+ qαqβ

rαβ

(1)





Neopentane C 380 0209 00Me 396 06061 00

Water Ow 3166 0646 minus 08476Hw +04238











2 3 4 5 6 7 8 9 10 11 12r (Aring)

-8

-6

-4

-2

0

2

4

6

8

10

12

14

PM

F (

kJm

ol)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa





1

2

3

4

5

6

ΔW (

kJ m

ol-1

)

01 200 400 600 800P (MPa)






Ka = 4π

int ra





System


01 443 441 438 420200 220 190 188 183400 209 167 167 153600 171 168 122 113800 111 123 107 091




nαβ = 4πρβ

int r2

r1

r2gαβ(r)dr (4)



0123456789

101112

g(r)

3 4 5 6 7 8 9 10 11r (Aring)

0123456789

101112

g(r)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa














νwt = nwNw

ntNt

minus 1 (5)






0

05

1

15

2

25

3

g(r)

0

05

1

15

2

25

3

g(r)

2 3 4 5 6 7 8 9 10 11 12r(Aring)

0

05

1

15

2

25

3

g(r)





0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

30

60

90

120

150

180

runn

ing

coor

d n

o

0

30

60

90

120

150

180

runn

ing

coor

d n

o


0

1

2

3

4

g(r)

0

1

2

3

4

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

1

2

3

4

g(r)








PW-W W-T T-W
















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)
























2 3 4 5 6 7 8 9 10 11 12r (Aring)

-8

-6

-4

-2

0

2

4

6

8

10

12

14

PM

F (

kJm

ol)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa





1

2

3

4

5

6

ΔW (

kJ m

ol-1

)

01 200 400 600 800P (MPa)






Ka = 4π

int ra





System


01 443 441 438 420200 220 190 188 183400 209 167 167 153600 171 168 122 113800 111 123 107 091




nαβ = 4πρβ

int r2

r1

r2gαβ(r)dr (4)



0123456789

101112

g(r)

3 4 5 6 7 8 9 10 11r (Aring)

0123456789

101112

g(r)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa














νwt = nwNw

ntNt

minus 1 (5)






0

05

1

15

2

25

3

g(r)

0

05

1

15

2

25

3

g(r)

2 3 4 5 6 7 8 9 10 11 12r(Aring)

0

05

1

15

2

25

3

g(r)





0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

30

60

90

120

150

180

runn

ing

coor

d n

o

0

30

60

90

120

150

180

runn

ing

coor

d n

o


0

1

2

3

4

g(r)

0

1

2

3

4

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

1

2

3

4

g(r)








PW-W W-T T-W
















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)



















Ka = 4π

int ra





System


01 443 441 438 420200 220 190 188 183400 209 167 167 153600 171 168 122 113800 111 123 107 091




nαβ = 4πρβ

int r2

r1

r2gαβ(r)dr (4)



0123456789

101112

g(r)

3 4 5 6 7 8 9 10 11r (Aring)

0123456789

101112

g(r)

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa

01 MPa

200 MPa

400 MPa

600 MPa

800 MPa














νwt = nwNw

ntNt

minus 1 (5)






0

05

1

15

2

25

3

g(r)

0

05

1

15

2

25

3

g(r)

2 3 4 5 6 7 8 9 10 11 12r(Aring)

0

05

1

15

2

25

3

g(r)





0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

30

60

90

120

150

180

runn

ing

coor

d n

o

0

30

60

90

120

150

180

runn

ing

coor

d n

o


0

1

2

3

4

g(r)

0

1

2

3

4

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

1

2

3

4

g(r)








PW-W W-T T-W
















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)



























νwt = nwNw

ntNt

minus 1 (5)






0

05

1

15

2

25

3

g(r)

0

05

1

15

2

25

3

g(r)

2 3 4 5 6 7 8 9 10 11 12r(Aring)

0

05

1

15

2

25

3

g(r)





0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

30

60

90

120

150

180

runn

ing

coor

d n

o

0

30

60

90

120

150

180

runn

ing

coor

d n

o


0

1

2

3

4

g(r)

0

1

2

3

4

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

1

2

3

4

g(r)








PW-W W-T T-W
















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)

















0

05

1

15

2

25

3

g(r)

0

05

1

15

2

25

3

g(r)

2 3 4 5 6 7 8 9 10 11 12r(Aring)

0

05

1

15

2

25

3

g(r)





0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

05

1

15

2

25

3

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

30

60

90

120

150

180

runn

ing

coor

d n

o

0

30

60

90

120

150

180

runn

ing

coor

d n

o


0

1

2

3

4

g(r)

0

1

2

3

4

g(r)

0 1 2 3 4 5 6 7 8 9 10r(Aring)

0

1

2

3

4

g(r)








PW-W W-T T-W
















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)





















PW-W W-T T-W
















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)



















HB nIIIHB EI

HB EIIHB EIII

HB τ IHB τ II

HB τ IIIHB








-3

-25

-2

-15

-1

-05

0

ln S

HB

Region I

-3

-25

-2

-15

-1

-05

0

ln S

HB

0 1 2 3 4time (ps)

-3

-25

-2

-15

-1

-05

0

ln S

HB

Region II

0 1 2 3 4time (ps)

Region III

0 1 2 3 4time (ps)




001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)

















001020304050607

f5


001020304050607

f4

001020304050607

f3

001020304050607

f2

01 200 400 600 800 01 200 400 600 800 01 200 400 600 800












ACKNOWLEDGMENTS






























don Oxford 1987)



















ACKNOWLEDGMENTS






























don Oxford 1987)
















The effect of aqueous solutions of trimethylamine-N-oxide on pressure induced modifications of...

Documents

Transcript of The effect of aqueous solutions of trimethylamine-N-oxide on pressure induced modifications of...