Báo cáo khoa học: Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents potx

Our recent molecular modeling stud-ies have depicted the molecular mechanism of the effects of different hydration percentages on the struc-tural [5] and enantioselective properties of e

and polar organic solvents

Nuno M Micaeˆlo and Cla´udio M Soares

Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, Portugal

The ability of enzymes to work in media other than

water is now widely accepted, and is the basis of

exten-sive basic research on enzyme catalysis and many

bio-technological applications [1] The fact that most

enzymes have evolved in an aqueous environment in

living cells does not mean that they cannot be

trans-ferred and be functional in a completely different kind

of medium [2–4] Our recent molecular modeling

stud-ies have depicted the molecular mechanism of the

effects of different hydration percentages on the

struc-tural [5] and enantioselective properties of enzymes [6]

when placed in organic solvents such as hexane Many

experimental studies in the field of nonaqueous

enzy-mology have focused their attention on demonstrating

that the amount of water in the organic medium plays

an important role in controlling the catalytic properties

of the enzymes [5–9] These studies have shown that when enzymes are used in organic solvents, water reta-ins its fundamental role in controlling the physical properties of the enzyme, and this role probably can-not be taken by other solvent In such systems, the effect of water is complicated to investigate, because this solvent is distributed in several phases; it can be in the vapor phase, adsorbed to the support material, dis-solved in the organic liquid phase, or bound to the enzyme [10]

Of the total water added to the organic medium, the effect of the organic solvents on the enzyme seems to

be primarily due to the water that is bound to the enzyme [7,11] This bound water is usually measured experimentally in terms of the thermodynamic activity

of water, assuming that, for enzymatic reactions

Keywords

enzyme hydration; organic solvents; protein

modeling; water clusters

Correspondence

C Soares, Instituto de Tecnologia Quı´mica

e Biolo´gica, Universidade Nova de Lisboa,

Av da Repu´blica, Apartado 127, 2781-901

Oeiras, Portugal

Fax: +351 21 4433644

Tel: +351 21 4469610

E-mail: claudio@itqb.unl.pt

Website: http://www.itqb.unl.pt/pm

(Received 21 December 2006, revised 1

March 2007, accepted 8 March 2007)

doi:10.1111/j.1742-4658.2007.05781.x

A comprehensive study of the hydration mechanism of an enzyme in non-aqueous media was done using molecular dynamics simulations in five organic solvents with different polarities, namely, hexane, 3-pentanone, diisopropyl ether, ethanol, and acetonitrile In these solvents, the serine protease cutinase from Fusarium solani pisi was increasingly hydrated with

12 different hydration levels ranging from 5% to 100% (w⁄ w) (weight of water⁄ weight of protein) The ability of organic solvents to ‘strip off’ water from the enzyme surface was clearly dependent on the nature of the organic solvent The rmsd of the enzyme from the crystal structure was shown to be lower at specific hydration levels, depending on the organic solvent used It was also shown that organic solvents determine the struc-ture and dynamics of water at the enzyme surface Nonpolar solvents enhance the formation of large clusters of water that are tightly bound to the enzyme, whereas water in polar organic solvents is fragmented in small clusters loosely bound to the enzyme surface Ions seem to play an import-ant role in the stabilization of exposed charged residues, mainly at low hydration levels A common feature is found for the preferential localiza-tion of water molecules at particular regions of the enzyme surface in all organic solvents: water seems to be localized at equivalent regions of the enzyme surface independently of the organic solvent employed

Abbreviations

FF, force field; MD ⁄ MM, molecular dynamics ⁄ molecular mechanics; SPC, single point change.

carried out in different media for a certain enzyme at

fixed water activity, the enzymes have equivalent

amounts of water bound [12] This approach to

expres-sing water content in organic solvents has been a

standard in nonaqueous enzymology, simplifying the

interpretation and prediction of changes in enzyme

performance It is commonly reported that the same

enzyme placed in different aqueous⁄ organic mixtures

with the same water activity has similar catalytic

con-stants This fact supports the idea that it is the

enzyme-bound water that modulates the catalytic

properties of the enzyme However, this relationship

does not hold for polar solvents, especially at high

water activity [10] It was hypothesized that solvents

such as alcohols are able to partially replace the role

of bound water, acting as ‘water replacers’ in

promo-ting enzyme activity [10] The failure of water activity

to predict the critical amount of water needed for

enzyme activity suggests that the organic solvent also

has a role in modulating the structure and dynamics of

the enzyme, probably by taking part in the solvation

mechanism of the enzyme It is clear that a concise

molecular picture of the solvation mechanism of

enzymes in nonaqueous solvents is needed

Reviewing what is known about protein hydration

takes us back to early protein hydration studies on

dry proteins There is a striking similarity between

the water-adsorption properties of proteins in air and

in organic solvents [13] Early studies of Yang &

Rupley [14] and Rupley et al [15], based on

calori-metric measurements of the heat capacity of the

lyso-zyme–water system, detailed the mechanism of the

hydration process of dry proteins The authors [14]

pointed out that the hydration steps for lysozyme

resemble the Hill model for the localized adsorption

of adsorbate onto a heterogeneous surface [16] They

interpreted the Hill model as follows: at low

cover-age, the adsorbate is dispersed on the surface; the

increase in adsorbate leads to the formation of a

con-densed phase of clusters; the clusters grow until the

surface is nearly covered and only the weakest sites

remain open; condensation of adsorbate over these

regions completes the adsorption process With this

model, Yang proposed that water clusters can be

viewed as mobile arrangements centered on polar

regions of the protein surface that increase in size

and number as water is added Protein–water

adsorp-tion isotherms in organic solvents and in the gas

phase [13] have shown that, at low water activity,

water adsorption by proteins suspended in nonpolar

organic solvents or by proteins equilibrated with

water from a gas phase are similar This has led to

the conclusion that the presence of an organic solvent

has little effect on the interaction between the protein and water in this water range

Parker et al [8] have detailed the mechanism of enzyme hydration (using subtilisin Carlsberg) in non-polar solvents using sensitive NMR experiments with deuterated water Their work clearly shows that in nonpolar solvents (hexane, toluene, and benzene) water

is preferentially localized in the most polar regions of the enzyme The majority of the enzyme surface is in direct contact with the organic solvent, and the forma-tion of a monolayer of water over the protein surface

is thermodynamically unfavorable However, no polar organic solvent was used in this study A similar study [17], using water sorption isotherms of lysozyme in nonpolar and polar organic solvents, previously sug-gested the same mechanism of protein hydration Io-nizable sites are hydrated first, followed by polar and nonpolar sites However, when the sorption isotherms

of toluene and n-propanol in the same water activity range are compared, they differ due to the competition

of the organic solvent for the enzyme hydration sites The hydration mechanism of enzymes in nonaque-ous solvents seems to be dictated by many factors, most of which have been addressed in the previous cited reports Not only do the properties of the organic solvent determine the relative partition of water at the enzyme surface at specific sites, but additionally, this solvent has a significant role in the solvation mechan-ism of the enzyme In this context, it would be import-ant for a molecular interpretation of the effects of the different quantities of enzyme-bound water in non-polar and non-polar organic solvents if the number of water molecules bound to the enzyme could be precisely measured, characterized, and localized [10,18] Our work is a comprehensive study of the hydration mech-anism of the enzyme cutinase in nonpolar (hexane, di-isopropyl ether, 3-pentanone) and polar (ethanol, acetonitrile) organic solvents with increasing hydration levels We have tried to determine how enzyme hydra-tion occurs in these media, and what the role is of the different organic solvents in the solvation mechanism

of the enzyme

Results and Discussion

Enzyme structure Protein molecular dynamics⁄ molecular mechanics (MD⁄ MM) simulations in organic solvents have been reported for several model systems [5,6,19–27] These simulation studies and the ones presented in this work typically involve one suspended isolated single protein molecule surrounded by water, ions, and organic

solvent Real nonaqueous experimental studies are

usu-ally carried on an immobilization support; however,

the system would be more complex if other factors,

such as protein aggregation, played an important role

in the protein function Our approach to the study of

protein structure and dynamics in such media has been

focused on understanding how proteins are affected by

the different hydration conditions when placed in

organic solvents [5,6] These studies have shown that

the structure, dynamics and enantioselectivity of

cu-tinase in hexane can be optimized within a specific

water hydration range [5,6,27] A more complete understanding of the structural properties of this enzyme in organic solvents is shown in Fig 1 Cu-tinase was simulated in five different organic solvents

of increasing dielectric constant with different hydra-tion condihydra-tions The water range studied for each solvent is a key aspect in understanding how the structural properties of the enzyme are modulated

by the hydration level Testing different organic sol-vents allows us to determine the role played by the organic solvent in the stabilization⁄ destabilization of

Fig 1 Average rmsd of Ca atoms of cutinase from the X-ray structure in (A) hexane, (B) diisopropyl ether, (C) 3-pentanone, (D) ethanol, and (E) acetonitrile, with different water percentages Calculations of rmsd deviations were done in the 3–7 ns period for each simulation and for all replicas Error bars are estimated from the SE of five to seven replicate simulations.

the enzyme structure The rmsd of the Ca atoms of

the protein fitted against the X-ray structure shows

that the enzyme structure is slightly different in each

organic solvent There are low rmsd values for the

enzyme in ethanol, and high values in acetonitrile For

hexane, diisopropyl ether, and 3-pentanone, the

enzyme Ca rmsd values at the different water

percent-ages are within 0.16 and 0.27 nm How do these data

compare with experimental data? The molecular

struc-tures of several enzymes soaked in organic solvents

have been determined by crystallographic studies

[28–34], but only limited changes in protein structure

were detected as compared with conventional aqueous

crystals Some authors argue that this approach could

hardly give any other answer, because if major

con-formational changes were to exist, it would be unlikely

that the crystal packing could be maintained [18]

Other methods are available that can provide

struc-tural information on these proteins in solution CD

studies of a-chymotrypsin in organic solvents have

shown a clear correlation between water content and

secondary structure of the enzyme [9,35] Fluorescence

measurements of enzymes in organic solvents have also

been used to investigate the structural changes of

enzymes Kijima and coworkers [36,37] have shown

that a-chymotrypsin enantioselectivity and fluorescence

properties are correlated with the solvent composition

These findings suggest a more dynamic picture of

enzyme structure rearrangement when enzymes are

placed in different organic solvents and have different

hydration levels The enzyme Ca rmsd measurement

from our simulations at different water percentages

and organic solvents show that different solvation

con-ditions yield different enzyme structural properties

This result is in agreement with the common

experi-mental observation that the solvent composition in

nonaqueous systems is able to affect enzyme structural

properties

Further analysis of the rmsd (Fig 1) suggests the

existence of minima in the rmsd data versus water

per-centage in the less polar organic solvents (hexane,

diisopropyl ether, and 3-pentanone) It is possible to

see that the structure of cutinase deviates less from the

X-ray structure when it is placed in hexane with 7.5%

water In the case of cutinase in diisopropyl ether, we

obtained the lowest rmsd at 30% water With a

slightly polar medium such as 3-pentanone, a local

rmsd minimum was observed at 40%; however, we

also observed low rmsd values at very low water

per-centages for this organic solvent It can be seen that

the optimal water content that minimizes the difference

from the X-ray structure is displaced to higher water

levels as we increase the polarity of the organic

solvent, as seen experimentaly [38] The dependence of enzyme structural properties on different water con-tents in organic solvents is a well-documented phenom-enon observed for several enzymes In some cases, a bell-shaped behavior of structural and biocatalytic properties is observed The rmsd data of our model enzyme placed in the less polar organic solvents with different hydration levels resemble this type of bell-shape behavior; this is clear in the case of hexane and diisopropyl ether However, this phenomenon is not clearly seen in our simulations in the case of polar, water-miscible organic solvents such as ethanol and acetonitrile This may be because only a small amount

of water is retained at the enzyme surface in the case

of polar organic solvents relative to nonpolar organic solvents, as detailed below

Water at the enzyme surface Spatial probability density

A key aspect of this work is the analysis of the local-ization of water at the enzyme surface We have suc-cessfully identified regions of high density of water in close contact with the protein for the different organic solvents tested In Fig 2, we show the spatial distribu-tion probability of water at 25% water content for the simulations in organic solvents and for the fully hydra-ted simulation This result was obtained by calculating the water probability density from all configurations of the last 3 ns for each organic solvent and for all repli-cates Probability densities were chosen in order to drawn contours at percentiles approximately between 93% and 98% for hexane, diisopropyl ether, and 3-pentanone, and at 99% for ethanol, acetonitrile, and water, giving the clearest picture for the preferential hydration regions near the enzyme surface From our simulations, we see that water does not partition appreciably to the organic solvent phase in the case of hexane, diisopropyl ether, and 3-pentanone, whereas in ethanol and acetonitrile, a considerable amount of water is found in the organic phase (results not shown) Note that in the fully hydrated system, we are also able to identify regions of higher density of water molecules

The water arrangement over the enzyme surface shows that there is no evidence of a complete water monolayer covering the enzyme (in fact, a monolayer would be possible at water percentages higher than 50%, although that never occurs), in accordance with previous suggestions that this would be thermodynam-ically unfavorable [8] The water distribution at the enzyme surface is clearly localized in certain regions, leaving other parts of the enzyme surface in direct

contact with the organic solvent This clearly suggests

that the role of the organic solvents should not be

undervalued, given that a significant enzyme surface

area is solvated by the organic solvent in all organic

solvents tested This means that water per se might not

account entirely for the solvation process of the

enzyme

An important discussion point that arises from our

studies is that the preferential binding sites of water at

the enzyme surface seem to be independent of the

organic solvent This is supported by the observation

that the water spatial probability distributions seem to

be equivalent for the same enzyme, regardless of the

organic solvent used (Fig 2) This appears to be true if

we look at Fig 2 and try to compare the spatial

prob-ability densities of water in hexane, diisopropyl ether,

and 3-pentanone They show that water is distributed

over similar regions of the enzyme in these three

sol-vents In the case of more polar solvents such as ethanol

and acetonitrile, we see that the water in these systems

is found in regions also present in the nonpolar solvents

As the enzyme surface does not change dramatically

when the enzyme is placed in different organic solvents,

water molecules seem to populate equivalent sites that

correspond to the areas of exposed charged⁄ polar side

chains hydrated to a higher or lower degree according

to the polarity of the organic solvent

Nonpolar organic solvents

In order to obtain a more precise picture of water at the surface of the protein, we looked at the number of water molecules within a specific layer of 0.25 nm from the surface of the protein (Fig 3), and the ratio of

Fig 2 Spatial distribution probability density

of water in (A) hexane, (B) diisopropyl ether, (C) 3-pentanone, (D) ethanol and (E) aceto-nitrile with 25% water and (F) in the fully hydrated system The molecular surface corresponds to the average structure of cu-tinase from the 3–7 ns period, for each sol-vation system and for all replicas For each organic solvent, two sides of the enzyme are shown in order to give a complete view

of the surface Each view of the enzyme has the same orientation in all organic sol-vents The contours enclose regions with a probability density above 9 · 10)6A˚)3for hexane, diisopropyl ether, and 3-pentanone,

4 · 10)6A˚)3for ethanol, 3 · 10)6A˚)3for acetonitrile, and 1 · 10)6A ˚ )3for the fully

hydrated system.

Fig 3 Average number of water molecules less than 0.25 nm away from the enzyme surface, for each organic solvent and water percentage Error bars are estimated from the SE of five to seven replicate simulations The total number of water molecules corres-ponding to each hydration level, for comparison with the number bound, is given in supplementary Table S3.

water to organic molecules in the region beyond this

layer (supplementary Table S1), for all organic solvents

and hydration levels This criterion is intended to

cap-ture the first layer of water molecules in direct contact

with the enzyme The first evidence from these curves

is the fact that they resemble the shape of the water

adsorption isotherms of enzymes in nonaqueous

sol-vents [13,17,39,40], although in these experimental

reports, water adsorption is plotted as a function of

water activity rather than water content These curves

show, first, a rapid increase in bound water, followed

by a second step in which there is a slow increase, and

then a third step of high water activity, where again

there is a sharp increase The water range that we

tes-ted seems to comprise the two initial steps Note that

the curves can easily discriminate nonpolar and polar

solvents In nonpolar solvents, water is highly retained

at the enzyme surface, whereas in polar solvents, water

is only weakly retained

In the particular case of hexane, most of the water

is located in this first hydration shell around the

enzyme, covering a large proportion of the surface

area of the enzyme but not achieving full coverage, as

seen in the previous section This organic solvent is the

one that allows the retention of the highest amount of

water at the enzyme surface The remaining water that

is not in direct contact with the enzyme is found in

secondary hydration layers Changing to a slightly

polar solvent such as diisopropyl ether, we see the

same trend for the water amount curve, but in this

case the amount of water in direct contact with the

enzyme is slightly lower, and it decreases even more as

the polarity of the organic solvent increases, as seen

for 3-pentanone This clearly suggests that, to obtain

the same amount of water bound to the enzyme, we

need to add more water to the system as we move to

more polar solvents The general trend for the amount

of water at the enzyme surface observed for these three

solvents shows that, at low water percentages, most of

the water present in the system is found at the surface

of the enzyme, and as water is added, it expands its

coverage over the enzyme surface up to a certain limit

For instance, it is possible to see that in diisopropyl

ether, the hydration of the enzyme surface reaches a

saturation point at about 40% water, corresponding to

90 water molecules at a distance less than 0.25 nm

from the enzyme surface For the case of 3-pentanone,

we see that at water percentages above 60%,

corres-ponding to 80 water molecules, there is almost no

more water retained at the enzyme surface In general,

it can be seen that as we change from apolar solvents

(hexane) to a slightly polar organic solvent

(3-penta-none), it becomes energetically favorable to have

organic solvent molecules instead of water molecules solvating the enzyme

Polar organic solvents

In water-miscible organic solvents such as ethanol and acetonitrile, the competition between the organic sol-vent and water for the enzyme surface is higher These two organic solvents can mimic the nonbonding prop-erties of water, and can effectively compete for the polar regions of the enzyme This is clearly seen in Fig 3, which shows that the amount of water bound

at the enzyme surface is very low as compared to the situation with nonpolar solvents In polar organic sol-vents, the amount of water bound to the enzyme increases slowly as water is introduced to the system, showing that, to obtain the same amount of water bound to the enzyme in nonpolar solvents such as hex-ane, it is necessary to add very high amounts of water The amount of organic solvent bound to the enzyme is very high, and the solvent clearly acts as a water re-placer in many polar and nonpolar regions of the enzyme surface This phenomenon clearly correlates with previous observations that polar organic solvents strip water from the enzyme surface to a higher extent than nonpolar ones [41] This result is also in agree-ment with the observation of McMinn et al [17] show-ing that in polar organic solvents, the amount of water bound to the enzyme at high water activities is signifi-cantly lower relative to the case when nonpolar organic solvents are used In this case, the organic sol-vent has a significant role in solvating the enzyme, and also takes part in the modulation of the structural and dynamic properties of the enzyme It is also of note that ethanol and acetonitrile are significantly different

in their ability to strip off water from the enzyme

In ethanol, at almost all hydration levels, we find twice

as much water bound to the enzyme as in aceto-nitrile; however, experiments show that more water is bound when the enzyme is equilibrated with a given water concentration in acetonitrile as compared with ethanol [17]

Water structure

In our simulated systems, we see that water molecules can be found in the bulk organic phase or at the enzyme surface In the water-miscible organic solvents,

we see a considerable amount of water in the organic solvent phase (results not shown) However, in all organic solvents, water seems to be organized in clus-ters at the enzyme surface (Fig 4A) These water clusters might have a substantial functional role in

modulating the structural and dynamic properties of

the enzyme, as suggested previously

In Fig 4A, we show the number of water clusters at

the enzyme surface A cluster is defined by two or

more water molecules at a minimum distance of

0.3 nm (distance between oxygen atoms) Note that

this criterion might not account for single water

mole-cules interacting with polar residues or water molemole-cules

solvating the ions in solutions (as oxygen atoms in this

case might be more than 0.3 nm distant) In Fig 4B,

we show the average number of water molecules per

cluster Again, the behavior in nonpolar and polar

sol-vents is easily distinguishable by this property In the

presence of nonpolar organic solvents, the number of

water clusters is almost identical in all hydration

con-ditions up to 25% (Fig 4A) In the nonpolar organic

solvents, the number of clusters grows rapidly as water

is added, indicating that water is being organized in

clusters of two or more water molecules at the enzyme surface, hydrating specific spots of the enzyme The number of clusters increases up to 25% water content, and beyond this water level, the number of clusters remains constant This indicates that, as water is added

to the system, new clusters of water are formed at available specific regions at the enzyme surface, which become fully occupied at 25% water content The size

of the clusters also grows as water is added, meaning that the water that is gradually introduced is also dis-tributed on pre-existing water clusters Water clusters

in nonpolar solvents are of similar size at low water percentages, but as water is added, the clusters in hex-ane become significantly larger than those in diisopro-pyl ether and 3-pentanone (Fig 4B)

In polar organic solvents, the water organization at the enzyme surface is different from that in the nonpo-lar organic solvents The cluster size increases more slowly as water is added, suggesting that most of the water introduced into the system is preferentially localized in the bulk organic solution The size of the clusters in ethanol and acetonitrile is fairly identical up

to 50% water content Above this level, water clusters

in ethanol are slightly larger that those in acetonitrile The molecular picture that arises from this analysis

of polar organic solvents is that water is largely frag-mented into single water molecules and small clusters

of water molecules around the protein In the case of nonpolar solvents, water is tightly bound to the enzyme and organized in clusters that grow in number and size in proportion to the water added

Ions in nonaqueous systems Ions play an important role in nonaqueous media, as they will allow the neutralization of exposed charged residues of the enzyme that cannot form intramolecu-lar ion pairs [42] This is evident from the X-ray struc-ture of trypsin in cyclohexane, which shows the existence of sulfate ions forming salt bridges or hydro-gen bonds with residues or water molecules [34] Work with different ion concentrations in nonaqueous system has also shown that ions have a marked impact on enzyme activity, relative to enzymes with no added salt [43–45]

In all organic solvent simulations, we have 10 Na+ and 10 Cl– docked to cutinase, as described in a previ-ous study [5] These ions neutralize individual charged groups at the enzyme surface We have seen that at equilibrium, water rearranges itself around the protein according to the type of the organic solvent used and the amount of water present in the system The same seems to be true for the ions In Fig 5, we analyze

Fig 4 (A) Average number of water clusters for each organic

sol-vent and water percentage (B) Average size of the water clusters

shown in (A) Error bars are estimated from the SE of five to seven

replicate simulations See text for details and comments regarding

this analysis.

how many charged residues are neutralized by sodium

(Fig 5A) and chloride (Fig 5B) counterions The first

evidence is that ions are not irreversibly bound to the

enzyme, as some charged residues initially stabilized by

ions are preferentially compensated by water

mole-cules, particularly at high water percentages Sodium

ions bound to the enzyme seem to be highly conserved

at low water percentages in all organic solvents As we

add water, sodium ions seem to be displaced from the

enzyme surface to the same extent in all organic

sol-vents These ions are found free in solution, hydrated

by water molecules, which, as we have seen, are

organ-ized in clusters Another common observation is the

formation of Na+Cl– ion pairs and, more rarely,

Na+Cl– tetrads in solution or at the enzyme surface,

where one of the negative or positive pair is a charged

residue Chloride ions are also displaced from the

enzyme surface by water in the same way as sodium

ions However it seems that more polar solvents such

as ethanol and acetonitrile, even at low water percent-ages, are able to replace chloride ions at the enzyme surface This phenomenon is more evident in acetonit-rile, as these chloride ions are rapidly removed from the enzyme, even at very low levels of hydration At high levels of hydration, only one ion or even none is found bound to the enzyme Another phenomenon seen in Fig 5A at low water percentages in nonpolar solvents is that some of the chloride ions stabilize more than one positive charged residue, as the 10 Cl– are in the proximity of more than 10 positive charged resi-dues What these results suggest is that ions can also

be ‘stripped off’ from the enzyme surface by the water molecules The charged residues initially stabilized by ions at low water percentages become preferentially compensated by water molecules as the water content increases This suggests that at low hydration levels, ions are important in the stabilization of charged resi-dues, but as the system becomes ‘more aqueous’, the exposed charged residues are preferentially stabilized

by water molecules The loss of the charge-counteract-ing effect provided by the ions near exposed charged residues could also be responsible for the structural changes observed at higher water percentages in all organic solvents

Water dynamics

We have seen that organic solvents with increasing polarity can structure, in different ways, the water at the enzyme surface It is also important to question how the dynamics of the water are modulated by the presence of the different organic solvents In a recent NMR study [46], the authors suggested a hydration model of subtilisin in tetrahydrofuran that comprised tightly bound, loosely bound and free water To ana-lyze the water dynamics at the enzyme surface, we recorded all the hydration events of all water mole-cules in the system A hydration event is the total time for which one water molecule is inside a layer of 0.25 nm around the enzyme surface The analysis was done during the last 3 ns of all trajectories for water contents of 25% and 60% All hydration events of all water molecules for a specific water content and organic solvent are collected and grouped in a fre-quency histogram For a clear analysis, the data were fitted using the Levenberg–Marquardt method to a two-exponential equation of the form:

fðxÞ ¼ a þ b  ecxþ d  efx where x stands for the time in ps that a water molecule

is inside a layer of 0.25 nm around the enzyme surface, and f(x) is the frequency of occurrence of that period

Fig 5 Average number of (A) negatively and (B) positively charged

residues neutralized by sodium and chloride ions, respectively, for

each organic solvent and water percentage Error bars are

estima-ted from the SE of five to seven replicate simulations.

of time Final parameters and standard errors are

shown in supplementary Table S2

The residence times of the water molecules at the

enzyme surface at 25% and 60% water content in the

different organic solvents and in the fully hydrated

sys-tem are shown on Fig 6 A general overview of this

figure indicates that many of the water molecules have

very low residence times at the enzyme surface

How-ever, a significant proportion of the water is retained

at the enzyme surface on a nanosecond time scale in

all organic solvents At 25% water content (Fig 6A),

it is possible to distinguish the effects of nonpolar and

polar organic solvents on the dynamics of the water at

the enzyme surface Water molecules in nonpolar

sol-vents are retained for longer periods of time at the

sur-face of the enzyme than in polar organic solvents

Particularly at 25% water content, the water residence

times in ethanol and acetonitrile are equivalent to

those in the pure aqueous system At 60% water

con-tent (Fig 6B), all four organic solvents (no data for

hexane at this water content) modulate the hydration

events in a progressive way; that is, water is retained for less time at the enzyme surface as the polarity of the organic solvent increases

It seems that, besides the differential structuring of water by the organic solvent, these water molecules organized in clusters at the enzyme surface do not behave as in a bulk aqueous solution Their dynamic properties, with respect to the residence time at the enzyme surface, are modulated by the polarity of the organic solvent

Concluding remarks

We have performed a systematic simulation study of the hydration mechanism of one enzyme in three dis-tinct classes of solvent: nonpolar organic solvents, polar organic solvents, and water We consider the effect of five different organic solvents, with different water percentages, on the structural properties of one model enzyme It is shown that the structural proper-ties of the enzyme in the less polar solvents (hexane, diisopropyl ether, and 3-pentanone) give a bell-shape curve, indicating that there is an optimum hydration level that allows the existence of a native-like structure

in solution This optimal hydration level for each organic solvent is obtained at increasing water percent-ages as we move to more polar solvents Our study also provides a detailed molecular picture of the hydration mechanism in the organic media, as indica-ted previously by experimental findings obtained by hydration studies in organic solvents by NMR and also from water adsorption experiments Our results show that water in nonaqueous media is organized at the enzyme surface in clusters of water molecules hydrating preferentially charged⁄ polar residues These clusters populate identical enzyme surface regions when the enzyme is placed in different organic sol-vents As water is added, these clusters grow in num-ber and size The nature of the organic solvent is able

to determine the size and number of clusters Nonpolar solvents allow the existence of large clusters of water molecules at the enzyme surface, whereas polar sol-vents fragment these clusters into smaller aggregates Polar solvents have the ability to replace water at some enzyme surface regions and contribute effectively to the structure and dynamics of the enzyme This means that water activity per se may not be sufficient to char-acterize the solvation of enzymes, and thus, water activity values in different organic solvent might not correlate directly with catalytic properties measured experimentally Ions seem to be preferentially bound

to the enzyme at low hydration levels Owing to the presence of the organic solvent, water is retained for

A

B

Fig 6 Water residence time frequency for each organic solvent

with (A) 25% water and (B) 60% water See text for details and

comments regarding this analysis.

longer at the enzyme surface, and this is more evident

in solvents with very low polarity On the other hand,

in high-polarity solvents, water at the enzyme surface

behaves similarly as in the fully hydrated system This

study has provided a detailed molecular picture of the

hydration mechanism of an enzyme, and shown it to

be clearly dependent on the nature of the organic

sol-vent and water content

Experimental procedures

Organic solvents force field

The organic solvents employed in this study were: hexane,

diisopropyl ether, 3-pentanone, ethanol, and acetonitrile

These organic solvents are commonly used in nonaqueous

enzymology studies, and are parameterized for MD⁄ MM

simulations Hexane was modeled as a flexible united atom

model using the gromos96 43a1 alkane parameters [47];

diisopropyl ether was taken from Stubbs et al [48] and

adapted to the gromos96 43a1 force-field (FF) [49,50];

3-pentanone is found in the gromos96 43a1 FF [51];

ethanol is also present in the gromos96 43a1 FF, and a

new parameterization of acetonitrile [52] was recently done

for this FF

These organic solvents have increasing dielectric

proper-ties and different partition coefficients [53] (Table 1) The

rationale for the choice of these organic solvents was to

have two groups with distinct properties, those that are

immiscible with water and that have low polar

characteris-tics (hexane, diisopropyl ether, and 3-pentanone), and those

that have polar properties and are water miscible (ethanol

and acetonitrile)

System setup

The general simulation methodology applied in the

MD⁄ MM simulations of cutinase in nonaqueous solvents

with increasing amounts of water was similar to the one

that we applied in a previous study [6] The 1.0 A˚

resolu-tion cutinase structure of Longhi was used [54], and the

protonated state of charged residues was estimated using a

methodology described previously [55] The selection of counterion positions and the different amounts of water hydrating the enzyme was done as explained in detail else-where [5] Five to seven replicates of 12 hydration levels were chosen, ranging from 5% to 100%; see supplementary Table S3 for a complete description of the molecular com-position of each system The replicates are composed of equivalent molecular systems (enzyme, ions, water, and organic solvent) with water molecules at slightly different positions at the enzyme surface Different hydration ranges were chosen for each organic solvent according to the knowledge that the critical amount of water that optimizes the structural and dynamic properties of the enzyme depends on the polarity of the organic solvent used [7] Each replicate of cutinase hydrated with a specific amount

of water was placed in a dodecahedral box with a minimum distance between the protein and box wall of 0.8 nm, and solvated with an equilibrated configuration of organic sol-vent molecules at 300 K Three replicate simulations of cutinase with ions in a fully hydrated system with single point change (SPC) water were also done

MD⁄ MM simulations

MD⁄ MM simulations were performed with the gromacs package [56,57] using the gromos96 43a1 FF [49,50] Bond lengths of the solute and organic solvent molecules were constrained with lincs [58], and those of water with settle [59] Nonbonded interactions were calculated using a twin-range method [50] with short-twin-range and long-twin-range cut-offs

of 8 A˚ and 14 A˚, respectively The SPC water model [60] was used in aqueous and in nonaqueous simulations A reaction field correction for electrostatic interactions [61,62] was applied, taking a dielectric of 54 for the fully hydrated system with SPC water [63] For the nonaqueous systems, the dielectric constant was chosen according to the experi-mental value reported in Table 1 The simulations were started in the canonical ensemble with initial velocities from

a Maxwell–Boltzmmann distribution at 300 K, and run for 50 ps with position restraints applied to all heavy atoms

of the protein and water molecules (force constant of

106kJÆmol)1Ænm)2) and a temperature coupling constant of 0.01 ps, allowing the equilibration of the organic solvent A further 50 ps of restrained simulation with the same force constant on the protein heavy atoms and temperature coup-ling constant was done for the equilibration of water mole-cules A final step of 50 ps was done with restraints only applied to the Ca carbons of the enzyme and a temperature coupling constant of 0.1 ps The unrestrained simulations were done in the isothermal–isobaric ensemble with an integration time step of 2 femtoseconds The protein, ions, organic solvent and water were coupled to four separated heat baths [64] with temperature coupling constants of 0.1 ps and a reference temperature of 300 K The pressure control [64] was implemented with a reference pressure of

Table 1 Dielectric constant and partition coefficient (log P) [53] of

the organic solvents employed in this work.

Dielectric constant (temperature K)

Partition coefficient (log P)

Diisopropyl ether 3.8 (303.2) 1.52