Destabilization of the Hydrogen-Bond Structure of Water by the

Jan 15, 2009 - ... yielding k1,NMR(c)/k1,NMR(0) ≈ 1.3 at 293 K. In ref 17, it was assumed that half of the water molecules are in the hydration shel...
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4038

J. Phys. Chem. B 2009, 113, 4038–4044

Destabilization of the Hydrogen-Bond Structure of Water by the Osmolyte Trimethylamine N-Oxide† Y. L. A. Rezus* and H. J. Bakker FOM-institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands ReceiVed: June 20, 2008; ReVised Manuscript ReceiVed: September 15, 2008

We use femtosecond mid-infrared pump-probe spectroscopy to investigate the effects of the osmolyte trimethylamine N-oxide (TMAO) on the structural dynamics of water. As a comparison, we also investigate the effects of other amphiphilic molecules: tetramethylurea (TMU), urea, proline, and N-methylacetamide (NMA). Our measurements show that TMAO has the unique property of increasing the orientational mobility of part of the water molecules in the solution, indicating that TMAO reorganizes the hydrogen-bond network of water in a special way. We also investigate the influence of the simultaneous presence in a solution of TMAO and urea, and of TMAO and NMA. It turns out that the effects of TMAO and urea are additive, whereas those of TMAO and NMA are nonadditive. I. Introduction The addition of small organic molecules, commonly known as osmolytes, to a protein solution dramatically affects the stability of the dissolved proteins. The effects depend on the nature of the osmolyte; that is to say, both stabilizing and destabilizing osmolytes exist. Urea, for example, induces proteins to unfold, while trimethylamine N-oxide (TMAO) is a powerful stabilizing osmolyte. This osmolyte not only counteracts the denaturing effects of urea but also protects proteins against thermal denaturation. Despite considerable research efforts, the detailed mechanism by which osmolytes affect protein stability has not yet been elucidated. Certain studies emphasize the role of direct interactions between osmolytes and the protein surface, whereas other studies suggest that the effect of osmolytes may be indirect. According to this latter view, osmolytes modify the hydrogen-bonding structure of the aqueous solvent, which alters the delicate balance that exists between the forces involved in protein folding (hydrophobic interactions, hydrogen bonds, etc.): as a result, the stability of the protein is modified. In this contribution, we use mid-infrared pump-probe spectroscopy to investigate the extent to which the hydrogenbond network of water is affected by the presence of osmolytes. Mid-infrared pump-probe spectroscopy provides information about the structural dynamics of water by probing the subpicosecond fluctuations of the hydrogen-bond network.1-7 In our experiments, we use the polarization-resolved version of the technique, which allows us to determine the reorientation time of water molecules. The orientational mobility of a water molecule can be interpreted as a measure of the rigidity of the hydrogen-bond network. This makes it possible to study the effects of osmolytes on the hydrogen-bonding structure of water by observing the changes that these solutes induce in the orientational dynamics of the water molecules. In the past, we have used mid-infrared pump-probe spectroscopy to study the orientational dynamics of water molecules in aqueous solutions of amphiphilic molecules.8,9 It was found that, as far as the orientational dynamics are concerned, two †

Part of the special section “Aqueous Solutions and Their Interfaces”. * To whom correspondence should be addressed. E-mail: [email protected].

types of water hydroxyl groups can be distinguished in these solutions. Part of the hydroxyl groups are relatively mobile in the sense that they display the same rapid orientational dynamics that are observed in pure liquid water (τrot ∼ 2.5 ps). In contrast, the remaining hydroxyl groups reorient on a much longer timescale (τrot ∼ 10 ps). We were able to show that these relatively immobile hydroxyl groups are involved in the solvation of the hydrophobic groups of the amphiphilic solutes. An interesting outcome of our experiments was that mobile hydroxyl groups (characterized by a bulk-like τrot of 2.5 ps) are observed even at extremely high solute concentrations (>10 mol/kg). This result was unexpected in view of the fact that the hydrogen-bond network of water is severely distorted at these concentrations as every water molecule is in contact with at least one solute molecule. Hence, although it is clear that these concentrated solutions do not in the strict sense contain any bulk water, part of the water hydroxyl groups do behave remarkably bulk-like. Here, we investigate how the orientational dynamics of the mobile water hydroxyl groups are affected by the presence of very high concentrations of organic solutes and osmolytes. The properties of such highly concentrated aqueous solutions are not only interesting from a physical-chemical perspective but also from a biochemical perspective. It is well-known that in the intracellular medium water is not present as a pure liquid but rather as a highly concentrated solution of organic molecules (macromolecules are present at concentrations as high as 400 g/L).10 Studying the properties of such highly concentrated solutions can therefore shed light on the properties of water molecules inside living organisms. II. Experimental Methods We have studied the orientational dynamics of water molecules in aqueous solutions of five amphiphilic molecules. These molecules include TMAO, urea, proline, tetramethylurea (TMU), and N-methylacetamide (NMA). To the water, we have added 2% heavy water (D2O), so that the aqueous solvent contains 4% HDO. The HDO molecules act as local probes whose rotational mobility can be determined with polarization-resolved pump-probe spectroscopy. The samples are placed between two CaF2 windows that are held apart by a Teflon spacer of 25 µm.

10.1021/jp805458p CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009

Effects of TMAO on the Structural Dynamics of Water

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Our laser system consists of a commercial Ti:sapphire regenerative amplifier that delivers 800 nm pulses with a duration of 100 fs and an energy of 1 mJ. About 70% of this light is used to pump an optical parametric amplifier (OPA) based on β -barium borate (BBO). The OPA is tuned to produce idler pulses with a wavelength of 2.0 µm. These pulses are frequency-doubled in a second BBO crystal. Subsequently, the pulses are difference-frequency mixed in a KNbO3 crystal, using the remaining 30% of the 800 nm light. This process yields mid-infrared pulses that are resonant with the OD-stretch vibration of HDO, having a duration of 150 fs, a wavelength of 4 µm, and an energy of a few microjoules. The mid-infrared light is coupled into a pump-probe setup. A small fraction of the light is split off by a wedged CaF2 window to obtain probe and reference pulses. The transmitted light forms the pump beam, and using a λ/2 plate, its polarization is set to 45° with respect to that of the probe beam. The pump, probe, and reference beams are focused onto the sample by an off-axis parabolic mirror and are recollimated by an identical mirror that is placed after the sample. The probe and reference beams are focused onto the entrance slit of a spectrometer, which disperses the beams onto a 2 × 32 liquid-nitrogen-cooled mercury-cadmium-telluride (MCT) array. Before entering the spectrometer, the probe beam passes through a polarizer allowing the selection of either its parallel or perpendicular polarization component with respect to the pump polarization. This selection results in transient absorptions ∆R|(ω, t) and ∆R⊥(ω, t), respectively. These two signals are initially different because of the preferential excitation of HDO molecules that have their OD groups aligned parallel to the pump polarization. As the delay between the pump and probe pulses is increased, molecular reorientation causes the molecules to lose memory of their initial orientation, and the difference between the two signals vanishes. The signals ∆R||(t) and ∆R⊥(t) can be combined to yield the anisotropy

R(t) )

∆R|(t) - ∆R⊥(t) ∆R|(t) + 2∆R⊥(t)

Figure 2. (left) Transient spectra of a 4 m solution of proline in isotopically diluted water (8% HDO in H2O). The dots represent the experimental data, and the solid lines are the fits to the relaxation model that is described in the text. (right) Delay scan of the data from the left part at a probe frequency of 2500 cm-1. The dots are the experimental data points; the solid line is the fit to the relaxation model; the dotted line represents the pure vibrational bleaching contribution to the transient signal, and the dashed-dotted line is the heating contribution.

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which is a quantity whose decay reflects the reorientation of the observed vibration. The isotropic signal

1 ∆Riso(t) ) (∆R||(t) + 2∆R⊥(t)) 3

Figure 1. Linear absorption spectrum of the OD stretch vibration of a solution of 4 M TMAO in 4% HDO:H2O (solid line) and of 4% HDO:H2O (dashed-dotted line).

(2)

is constructed in such a way that it is free of reorientational processes and reflects the decay of the excitation by vibrational relaxation. III. Results and Discussion A. Isotropic Data. In Figure 1, the linear absorption of the OD stretch vibration of a 6 M solution of TMAO in HDO:H2O is compared with that of the pure HDO:H2O solvent. It is seen that the addition of the amphiphilic molecules has very little effect on the shape and position of the linear OD stretch absorption spectrum. This means that the addition of these molecules does not lead to a strong change in the strength of the hydrogen-bond interactions between the water molecules. Figure 2 displays the transient absorption changes that are observed for a 4 m solution of proline in water. The isotropic signal decays on a 2 ps time scale, which is typical for the vibrational energy relaxation of the OD vibration of HDO.7,11 At long delays, the sample exhibits a residual absorption change

that does not decay on the time scale of our experiment (∼500 ps). This persistent transient signal results from the temperature rise that occurs in the sample once the energy of the pump pulse thermalizes.7,12 Upon heating, the OD band shifts to higher frequencies, which results in the particular spectral signature of the long-time signal, i.e., a bleach on the low-frequency side of the spectrum and an induced absorption on the high-frequency side. The vibrational relaxation dynamics of HDO molecules have been extensively investigated in different types of solutions.1,7,9,11 It has been well established that the relaxation proceeds in a two-step fashion, as is illustrated in Figure 3. First, the energy of the OD vibration is transferred to an intermediate state (0*). This state should be viewed as a state in which one or more low-frequency modes that couple anharmonically to the OD vibration become populated. The second step in the relaxation is the decay of the intermediate level to the heated ground state mentioned in the previous paragraph. It should be noted that we do not directly observe the intermediate state but only the effect of the occupation of this state on the frequency and cross section of the initially excited OD-stretch vibration. In an earlier study, we have shown that this relaxation model leads to the following expression for the transient absorption of the OD vibration of HDO9

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Rezus and Bakker

∆R(ω, t)/N1(0) ) {σ12(ω) - 2σ01(ω)}e-k1t + k* k1 e-k*t e-k1t + 1 + ∆σ01(ω) k* - k1 k* - k1 k1 (e-k1t - e-k*t) (3) ∆σ*01(ω) k* - k1

{

}

* * where ∆σ01 ) σ01 - σ01 and ∆σ01 ) σ01 - σ01; k1 and k* are the inverse lifetimes of the excited state and the intermediate state, respectively. We have fit this model to the data in Figure 2. We see that the model provides an excellent description of the data. The same model has been used to describe the relaxation dynamics at other proline concentrations. From these fits, we have obtained the lifetimes of the levels as a function of the proline concentration (Figure 4, left). Both lifetimes show a slight increase as the proline concentration is increased. The same conclusion can be drawn for solutions of TMAO in water. The time-dependent transient signal is excellently described by the relaxation model. When we regard the concentration dependence of the lifetimes of the two states, we observe a similar increase as was observed for proline (Figure 4, right). B. Anisotropic Data. The relaxation model described above allows us to decompose the measured transient signal into a signal that purely reflects the vibrational dynamics of the OD vibration and a heating signal (Figure 2, right). As has been pointed out in previous work, only the pure vibrational signal should be used to calculate the anisotropy.7,9,12 The intermediate state, or rather the effect of the intermediate on the frequency and cross section of the OD vibration, shows the same anisotropy as the initially excited OD vibration. Therefore, the anisotropy dynamics of the OD vibration can be obtained by subtracting only the heating contribution from the raw transient signal and by computing the anisotropy using this corrected signal. Figure 5 displays a number of thus obtained anisotropy decay curves for solutions of NMA, proline, and TMAO. The extrapolated initial value of the anisotropy is close to 0.4. This indicates that the contribution of fast inertial (librational) motions to the anisotropy decay must be small. For pure water, the anisotropy is known to decay monoexponentially with a time constant of 2.5 ps,1,7 as can also be judged from Figure 5, left. As soon as a solute is added, however, the anisotropy decay becomes biexponential, with a fast component (τrot e 2.5 ps) and a much slower component. In general, the observation of biexponential relaxation can have two causes: it is either the effect of an intrinsic biexponential relaxation mechanism, or it is caused by the presence of two populations showing distinctly different dynamics.13 Intrinsic biexponential relaxation can be described with a wobbling-ina-cone model.14,15 In this model, the water molecules show a fast reorientation within a limited cone angle and a slower reorientation due to the orientational diffusion of the cone over the whole angular space. However, for the description of the presently observed biexponential dynamics, a single wobblingin-a-cone model is inappropriate, because such a description implies that all water molecules show the same (nonexponential) orientational dynamics. However, an aqueous solution constitutes a strongly inhomogeneous system, and one expects different water molecules to show different dynamics. This latter picture is also supported by molecular dynamics simulations of salt solutions that show that the orientational dynamics of water molecules in the hydration shell differ from the dynamics of the bulk water molecules.16 We observe that the amplitude of the slow component scales linearly with the solute concentration (Figure 6). This observa-

Figure 3. Model used to describe the relaxation of the OD vibration of the HDO molecule in aqueous solutions of different molecules. The excited state first decays to an intermediate state that has a different spectrum than the ground state. The subsequent decay of this level leads to thermalization of the vibrational energy and, as such, to heating of the sample. In this figure N1, N0, N*0, and N0′ represent the populations of the levels and σ01, σ12, σ01, and σ01 the cross sections.

Figure 4. Concentration dependence of the lifetimes of the excited state (circles) and of the intermediate state (squares) of the OD vibration of HDO for solutions of proline (left) and TMAO (right).

tion strongly supports an assignment of the slow component to water hydroxyl groups that are part of the solvation shell of the solute molecule. Hence, the biexponential relaxation behavior originates from the presence of two distinct populations of water hydroxyl groups: hydroxyl groups in the solvation shell showing slow orientational dynamics and hydroxyl groups outside this shell showing bulk-like orientational dynamics. In a previous publication, it was found that the slow hydroxyl groups are part of the solvation shell of the hydrophobic part of the amphiphilic solutes.8 In the following we shall, for the sake of brevity, refer to these two fractions of water hydroxyl groups as the mobile and the immobile fractions. At concentrations >5 m, the relation between the amplitude and the concentration is no longer linear, indicating that the solutes start to share their solvation shell. Figure 7a compares the fast components in the anisotropy decay of two NMA solutions. We clearly see that the reorientation time of the mobile water fraction is identical for these two NMA concentrations. Figure 7b shows a plot of the reorientation time of the fast component versus the solute concentration for solutions of NMA. It appears that the reorientation time of the mobile water fraction is completely unaffected by the NMA concentration, up to the highest concentration used (8 mol/kg). This is unexpected since at this concentration all water molecules are in direct contact with at least one NMA molecule. Apparently the mobile hydroxyl groups are unaffected in their reorientation, even though they are in close contact with an NMA molecule. For aqueous solutions of TMAO, a different situation occurs (Figure 8). In these solutions, we observe a decrease of the reorientation time of the mobile water fraction with increasing TMAO concentration. This behavior of TMAO is unique among the studied solutes, as is shown in Figure 9. This figure displays the reorientation time of the mobile water fraction as a function of the solute concentration for five different solutes, one of which is TMAO. We see that TMAO is the only solute that affects the reorientation time of the mobile water

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Figure 5. Anisotropy decays for the center of the OD band (νprobe ) 2500 cm- 1) for aqueous solutions of NMA (left), proline (middle), and TMAO (right) at varying concentrations. The solid lines represent fits to a monoexponential with an end level (R(t) ) (A0 - A∞) exp(- t/τrot) + A∞).

Figure 6. End level in the anisotropy as a function of the concentration of solute for aqueous solutions of NMA (left), proline (middle), and TMAO (right).

Figure 7. (a) Logarithmic plot of the fast component in the anisotropy decay for 2 and 8 m NMA solutions. (b) Reorientation time constant of the mobile water fraction in aqueous NMA as a function of the NMA concentration. The decays are constructed by subtracting the end level (as obtained from the fit) from the original anisotropy decay and normalizing the result to unity.

Figure 8. (a) Logarithmic plot of the fast component in the anisotropy decay for 2 and 8 m TMAO solutions. (b) Reorientation time constant of the mobile water fraction in aqueous TMAO as a function of the TMAO concentration. The decays are constructed by subtracting the end level (as obtained from the fit) from the original anisotropy decay and normalizing the result to unity.

fraction; for the other four solute molecules, the reorientation time of the mobile water molecules is 2.5 ps, identical to the value observed in pure water. The measurements discussed above make it clear that, in comparison to other amphiphilic solutes, TMAO has a unique effect on the orientational dynamics of the mobile water fraction. We observe that the time constant of this mobile fraction strongly decreases with increasing TMAO concentration. As a next experiment, it would be interesting to investigate the effect of the simultaneous presence in a solution of TMAO and a second solute. We have carried out this experiment with urea and NMA as the secondary solutes. Figure 10a shows the reorientation time of the mobile water fraction for solutions that contain 6 m TMAO in addition to varying concentrations of urea. We see that the effect of TMAO, which consists of speeding up the reorientation of the mobile water fraction, is reversed by adding urea to the solution. A different situation occurs, however, when we consider solutions in which TMAO is simultaneously present with NMA (Figure 10b). For these solutions, we observe that the effect of TMAO on the reorienta-

tion time of the mobile water fraction is enhanced by NMA: the reorientation time becomes shorter upon the addition of NMA. We note that NMA alone did not affect the reorientation time of the mobile water fraction. IV. Discussion The vibrational relaxation of the OD vibration of HDO in H2O proceeds via a two-step mechanism.7 Here, we have shown that this relaxation model remains valid at very high solute concentrations. Apparently the relaxation mechanism of the OD vibration is not altered by the presence of amphiphilic solutes: only the relaxation rate is affected. In general, this rate decreases with increasing solute concentration. However, the dependence is very weak, as can be judged from the fact that the lifetimes of the two levels increase by as little as 25% in a 10 m solution of TMAO. The slowing down of the relaxation does not seem to depend on the nature of the amphiphilic solute. Therefore, the slight slowing down of the population relaxation dynamics appears to be caused by the truncation of the hydrogen-bond network due to the presence of the solute molecules.

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Figure 9. Reorientation time constants of the bulk water fraction for different osmolyte solutions as a function of the osmolyte concentration: tetramethylurea (TMU), urea, N-methylacetamide (NMA), proline, and trimethylamine N-oxide (TMAO).

Rezus and Bakker of the other (no longer observed) water molecules in the solution. It should be noted that this bias for the dynamics of the hydration shells at longer times is not present in this work. For the organic solutes studied here, the vibrational lifetime of the water molecules in the solvation shells is the same as that of the water molecules in the bulk. The data shown in Figures 4 and 5 show that part of the water hydroxyl groups in amphiphilic solutions display considerably slower orientational dynamics than in pure water. It is interesting to compare these findings to results of NMR studies. There have been several NMR studies of the effects of hydrophobic solutes on the reorientation of water molecules.17-19 These studies make use of the fact that the reorientation of the water molecule dipoles generates the fluctuations required for the longitudinal spin relaxation. This effect is assumed to be in the motional narrowing limit. In relating the spin relaxation time to the reorientation time of the solvation shell, it is assumed that only the reorientational dynamics in the hydration shell are affected and that the observed relaxation is an average of the molecules in the solvation shells and the water molecules outside these shells. Hence, the effect of the solute on the spin relaxation is described with the following equation:

k1,NMR(c) (COH - nc) nc τrot,s + ) k1,NMR(0) COH COH τrot,0

Figure 10. (a) Reorientation time of the mobile water fraction in solutions that contain varying concentrations of urea (open squares) and in solutions that contain varying concentrations of urea in addition to 6 m TMAO (filled circles). (b) Reorientation time of the mobile water fraction in solutions that contain varying concentrations of NMA (open squares) and in solutions that contain varying concentrations of urea in addition to 6 m NMA (filled circles).

For all amphiphilic solutes and at all solute concentrations, the anisotropy shows a biexponential decay that results from the presence of two distinct types of water hydroxyl groups: hydroxyl groups in the hydration shell of the hydrophobic groups of the solute and hydroxyl groups outside these shells. This twocomponent picture agrees with the results of previous molecular dynamics simulations16 and earlier femtosecond mid-infrared studies of the dynamics of water in solutions containing Cl-, Br-, and I- ions.13 In these studies, it was observed that the dynamics of the water hydroxyl groups solvating the anions differ from the dynamics of the bulk.16,13 In a more recent femtosecond mid-infrared study of the orientational dynamics of water in solutions of Br-, it was assumed that the measurements represent the dynamics of all water molecules in solution at all delay times.14,15 This assumption is not necessarily valid, because the water hydroxyl groups that form a hydrogen bond to Cl-, Br-, and I- show a much longer vibrational lifetime than the water hydroxyl groups that form hydrogen bonds to an oxygen atom of another water molecule.13-15 Due to the difference in lifetime, after a few picoseconds, the nonlinear absorption signal of the water molecules outside the hydration shells has disappeared, and the observed signal (and its anisotropy) is only due to the water hydroxyl groups that are hydrogen bonded to the anions. Hence, at later delay times, only the orientational dynamics of the water hydroxyl groups residing in the first hydration shell of the ion are observed,13 and these dynamics can be very different from the orientational motions

(4)

with k1,NMR(c) being the spin relaxation rate at solute concentration c, COH the concentration of OH groups in water (110 mol/ kg), n the number of OH groups per solute molecule showing τrot,s, τrot,s the reorientation time of water in the solvation shell, and τrot,0 the reorientation time of bulk liquid water. In all NMR studies, it is observed that k1,NMR(c) increases with increasing number of methyl groups in the solute,17-19 which shows that the reorientation slows down for water molecules solvating the hydrophobic part of the molecule. In a study of solutions of tertiary butyl alcohol (TBA) (for which the hydrophobic part is comparable to TMAO), it was found that k1,NMR(c)/k1,NMR(0) ) 1.375 at 29.9 °C and 1.625 at 7.7 °C.17 Interpolation gives a ratio of k1,NMR(c)/k1,NMR(0) ) ∼1.5 at 293 K. In ref 20, the effect of TMAO on the average reorientation was measured, yielding k1,NMR(c)/k1,NMR(0) ≈ 1.3 at 293 K. In ref 17, it was assumed that half of the water molecules are in the hydration shell of TBA, from which it would follow that τrot,s/τrot,0 ≈ 2 at room temperature. As τrot,0 of bulk water is ∼2.5 ps, τrot,s would thus be ∼5 ps. However, as NMR only probes the aVerage reorientation dynamics of all water molecules, the observed change in k1,NMR can result equally well from a large number of moderately affected hydroxyl groups as from a much smaller number of strongly affected hydroxyl groups. Therefore, there is an ambiguity in the values of n and τrot,s that follow from NMR data. The femtosecond measurements suffer from another problem. As these measurements probe the anisotropy over a limited time range, these measurements only give a lower boundary for τrot,s. It is possible to estimate values for τrot,s and n by combining the NMR and the femtosecond data. We can fit the data shown for TMAO in Figure 4 to a combination of n and τrot,s using as a boundary condition that the average orientation time of all water molecules as observed with NMR increases by a factor of 1.3-1.5. From this fit, it follows that τrot,s would be in the range from 8 ps (with n ) 19) to 12 ps (with n ) 15). Here, it should be noted that the quality of the fit of the femtosecond data increases with increasing value of τrot,s, especially for the higher solute concentrations (>4 M). In

Effects of TMAO on the Structural Dynamics of Water ref 19, an interesting suggestion is made concerning the precise origin of the slow component. It is proposed that the slow component is not due to n water hydroxyl groups showing an exclusively slow reorientation but due to m > n hydroxyl groups in the solvation shell showing a partial fast and a partial slow reorientation. This can indeed not be excluded, as the femtosecond measurements only provide the net contribution of all solvating water molecules to the overall observed water orientational dynamics. The slowing down of the reorientation of the hydroxyl groups near the hydrophobic part of the solute can be understood by considering the reorientation mechanism of water.20,21 Liquid water is a highly coordinated system in which most water molecules are tightly hydrogen-bonded to four neighbors. In such a network, the water molecules cannot reorient via the complete breaking and reforming of hydrogen bonds with new partners, since the dissociation energy of a hydrogen bond lies far above the thermal energy available to a water molecule. Instead, reorientation is facilitated by the presence of network defects, i.e., five-coordinated water molecules.20,21 Such fivecoordinated water molecules are characterized by the presence of a bifurcated hydrogen bond, in which an OH group is simultaneously bound to two other water molecules. A bifurcated hydrogen-bond configuration allows for the concerted breaking and reformation of a hydrogen bond, thereby considerably lowering the activation energy for reorientation.21,22 The creation of 5-fold coordinated water molecules requires a certain amount of space. Around hydrophobic groups, for instance, methyl groups, this space is not available. Therefore, the immobilization of water hydroxyl groups by hydrophobic groups is likely the consequence of a steric effect, in which the hydrophobic group prevents a hydrogen-bonding partner from approaching a water molecule that is already tetrahedrally coordinated. An open question concerns the behavior of the OD groups that are hydrogen-bonded to the hydrophilic groups of the amphiphilic solutes. Because there are not that many of these OD groups (two or three per solute molecule), it is not possible to identify these OD groups in the observed anisotropy dynamics. On the basis of earlier results obtained for solutions of urea in water,5 it is to be expected that the OD groups solvating the hydrophilic groups show dynamics that are not very different from bulk water. In this case, these OD groups would be contained in the mobile fraction of the water molecules. The TMAO solutions display a unique behavior in comparison to the other solutions. In the case of TMAO as a solute, the reorientation time of the mobile water fraction is no longer independent of the solute concentration. Rather, the reorientation time decreases as the TMAO concentration is increased. The question arises as to what the physical mechanism may be that leads to these faster orientational dynamics. It is likely that the polar NO group of the TMAO molecule would be involved. A possible explanation could be that the NO group increases the local density of network defects (i.e., 5-fold coordinated water molecules) because of its poor fitting in the hydrogen-bond network of water. The oxygen atom of TMAO contains three lone pairs which can each accept a hydrogen bond. This is in contrast with the two hydrogen bonds that can be accepted by the oxygen atom of a water molecule. It is reasonable to assume that oxygen atoms that can accept more than two hydrogen bonds will disrupt the tetrahedral hydrogen-bond network of water. This disruption will result in a higher concentration of network defects, i.e., five-coordinated water molecules, and in

J. Phys. Chem. B, Vol. 113, No. 13, 2009 4043 view of the mechanism of reorientation of liquid water, to a faster reorientation. Clearly, at this point, this is only a possible explanation. We hope that the present observations will stimulate theoretical work to find the origin of the acceleration of the reorientation of the mobile water fraction by TMAO. We have also considered the effects of combinations of two different solutes on the dynamics of the mobile water fraction. In particular, two such combinations were investigated: TMAO with urea and TMAO with NMA. The former combination is interesting because of the opposite effects of urea and TMAO on the stability of proteins. The interest of the latter combination lies in the fact that NMA forms a model for the protein backbone, since it contains the amide motif (CONH). In fact, NMA constitutes a better model for the protein backbone than do free amino acids, since these are generally present as zwitterions. The results on the solutions of TMAO and urea show that the effects of these two solutes are additive. Adding urea to a solution of TMAO causes the reorientation time of the mobile fraction to increase back to the value observed in pure water. In contrast, for TMAO and NMA, we observe a completely different picture. While NMA alone does not affect the dynamics of the mobile fraction, the addition of NMA to a solution that already contains TMAO leads to a further decrease in the reorientation time of the mobile water fraction. This shows that the effects of TMAO and NMA are nonadditive. Following our line of thought from the previous paragraph, we conclude that the addition of NMA to a solution of TMAO increases the concentration of network defects even beyond the concentration present in a solution containing only TMAO. It thus seems that, in the absence of TMAO, amide groups can be accommodated by the hydrogen-bond network without the creation of network defects, while, in the presence of TMAO, NMA can only be solvated if network defects are created. This could indicate that TMAO modifies the hydrogen-bond network in such a way that the solvation of amide groups is hampered, which would in turn provide a clue as to how TMAO stabilizes proteins. We end the discussion of our results by considering what the role of the mobile water fraction may be in biological systems. As we have seen that the mobile water fraction persists up to very high solute concentrations, it is reasonable to assume that a similar mobile water fraction will also exist in the intracellular medium. It is well-known that solvent fluctuations play an important role in the functioning of proteins and other biomolecules.10 Therefore, it would be tempting to speculate that it is the presence of this mobile water fraction which allows biomolecules to retain their functionality inside the crowded interior of cells. V. Conclusions We have studied the effects of TMAO and other amphiphilic solutes on the vibrational relaxation and the orientational dynamics of HDO molecules in water. The vibrational relaxation rate of the OD vibration is generally seen to decrease as the solute concentration is increased. The effect is not very strong: in a 10 m solution of trimethylamine N-oxide (TMAO), for example, the relaxation of the OD vibration of HDO proceeds only 25% slower than in pure water. As far as the orientational dynamics are concerned, two types of water hydroxyl groups can be distinguished in these solutions: relatively mobile water hydroxyl groups whose reorientation time is similar to the value observed in pure water (τrot,0 ) 2.5 ps) and relatively immobile water hydroxyl groups that are involved in the solvation of the hydrophobic groups of the amphiphilic solutes.

4044 J. Phys. Chem. B, Vol. 113, No. 13, 2009 For most solutes, the reorientation time of the mobile water fraction is 2.5 ps and this value is independent of the solute concentration. TMAO, on the other hand, displays the unique property of increasing the reorientation rate of the mobile water fraction. This is explained by assuming that TMAO increases the density of network defects in the hydrogen-bond network of water. We have also investigated the effects of the simultaneous presence of TMAO and urea and of TMAO and N-methylacetamide (NMA) on the reorientation rate of the mobile water fraction. The effects of TMAO and urea are additive, while those of TMAO and NMA are nonadditive. Acknowledgment. This work is part of the research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)”, which is financially supported by the “Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO)”. We would like to thank Damien Laage and Casey Hynes for useful discussions. References and Notes (1) Tan, H. S.; Piletic, I. R.; Fayer, M. D. J. Chem. Phys. 2005, 122, 174501. (2) Cringus, D.; Yeremenko, S.; Pshenichnikov, M. S.; Wiersma, D. A. J. Phys. Chem. B 2004, 108, 10376.

Rezus and Bakker (3) Dokter, A. M.; Woutersen, S.; Bakker, H. J. Phys. ReV. Lett. 2005, 94, 178301. (4) Fecko, C. J.; Eaves, J. D.; Loparo, J. J.; Tokmakoff, A.; Geissler, P. L. Science 2003, 301, 1698. (5) Rezus, Y. L. A.; Bakker, H. J. Proc. Natl. Acad. Sci. U.S.A 2006, 103, 18417. (6) Woutersen, S.; Bakker, H. J. Nature 1999, 402, 507. (7) Rezus, Y. L. A.; Bakker, H. J. J. Chem. Phys. 2005, 123, 114502. (8) Rezus, Y. L. A.; Bakker, H. J. Phys. ReV. Lett. 2007, 99, 148301. (9) Rezus, Y. L. A.; Bakker, H. J. J. Phys. Chem. A 2008, 112, 2355. (10) Ball, P. Chem. ReV. 2008, 108, 74. (11) Piletic, I. R.; Moilanen, D. E.; Spry, D. B.; Levinger, N. E.; Fayer, M. D. J. Phys. Chem. A 2006, 110, 4985. (12) Steinel, T.; Asbury, J. B.; Zheng, J.; Fayer, M. D. J. Phys. Chem. A 2004, 108, 10957. (13) Kropman, M. F.; Nienhuys, H.-K.; Bakker, H. J. Phys. ReV. Lett. 2002, 88, 077601-1.. (14) Park, S.; Fayer, M. D. Proc. Nat. Acad. Sci. U.S.A. 2007, 104, 16731. (15) Park, S.; Moilanen, D. E.; Fayer, M. D. J. Phys. Chem. B 2008, 112, 5279. (16) Laage, D.; Hynes, J. T. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 11167. (17) Yoshida, K.; Ibuki, K.; Ueno, M. J. Chem. Phys. 1998, 108, 1360. (18) Shimizu, A.; Fumino, K.; Yukiyasu, K.; Taniguchi, Y. J. Mol. Liq. 2000, 85, 269. (19) Qvist, J.; Halle, B. J. Am. Chem. Soc. 2008, 130, 10345. (20) Laage, D.; Hynes, J. T. Science 2006, 311, 832. (21) Sciortino, F.; Geiger, A.; Stanley, H. E. Nature 1991, 354, 218. (22) Gallagher, K. R.; Sharp, K. A. J. Am. Chem. Soc. 2003, 125, 9853.

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