7932
2007, 111, 7932-7933 Published on Web 06/20/2007
The Influence of Urea and Trimethylamine-N-oxide on Hydrophobic Interactions Sandip Paul and G. N. Patey Department of Chemistry, UniVersity of British Columbia, VancouVer BC V6T 1Z1, Canada ReceiVed: May 2, 2007
Molecular dynamics simulations are used to obtain potentials of mean force for pairs of neopentane molecules immersed in aqueous solutions containing urea, trimethylamine-N-oxide (TMAO), or both solutes at once. It is shown that the hydrophobic attraction acting between neopentane pairs in pure water and in water-urea solution is completely destroyed by the addition of TMAO. This strongly suggests that TMAO does not counter the protein denaturing effect of urea by enhancing hydrophobic attraction amongst nonpolar groups.
Hydrophobic interactions among nonpolar groups are believed to be a significant factor contributing to the stability of folded proteins in aqueous solution.1 In urea solution (∼8 M), folded states become unstable with respect to more expanded configurations, leading to so-called chemical denaturation of proteins. Interestingly, if trimethylamine-N-oxide (TMAO) (∼4 M) is added to the water-urea-protein system, it acts to stablize the folded state, countering the denaturing effect of urea.1 The mechanism of urea-induced protein denaturation is not fully understood and remains a subject of active research. Nevertheless, the available evidence suggests that urea denaturation likely occurs through some combination of two possible pathways. Urea can act indirectly, altering the water structure, thereby reducing hydrophobic interactions, or directly through hydrogen bonding with the peptide backbone.1-4 The mechanism by which TMAO counters the denaturing effect of urea is also not completely understood. It has been suggested1,5 that the main effect comes through TMAO-induced alterations of water-water structure and interactions, but we have recently argued that this is unlikely.6 Rather, we have suggested6 that a possible mechanism might simply be preferential solvation; both water and urea interact strongly with TMAO (through hydrogen bonds), and we would expect this to reduce their ability to solvate the protein. However, another possibility is that TMAO acts to increase hydrophobic attractions, hence, stabilizing the folded state. The purpose of the present paper is to examine this possibility. Previous simulations have shown that the influence of urea on the hydrophobic interactions of nonpolar solutes depends on the solute size7-9 and, to some extent, on the models employed.10 For methane modeled as a simple Lennard-Jones (LJ) particle, urea is actually found to strengthen the hydrophobic interaction, increasing the stability of the contact pair.7,9 On the other hand, for neopentane (modeled as a larger LJ particle), urea can weaken the hydrophobic attraction, reducing the stability of the contact pair.9,10 However, Lee and van der Vegt have recently reported10 that the influence of urea on the neopentane-neopentane potential of mean force (PMF) depends * Corresponding author. E-mail:
[email protected].
10.1021/jp0733668 CCC: $37.00
qualitatively on the molecular models employed, and simple generalizations are not possible. Some of the results discussed below serve to further emphasize this point. Athawale et al.11 have calculated methane-methane potentials of mean force for TMAO solutions up to 3 M in concentration. They conclude that TMAO has negligible effect on the effective methanemethane interaction. Because the effects of both urea and TMAO have been shown to be small for methane pairs, in this paper, we focus on the neopentane case. The water, urea, and TMAO molecules are modeled as described in ref 6. Following Shimizu and Chan,9 neopentane is modeled as a LJ sphere with the parameters given by Kuharski and Rossky12 ( ) 3.496 kJ mol-1, σ ) 6.15 Å). Our objective is to determine how the neopentane-neopentane PMF is altered by addition of TMAO. For example, if TMAO were to give a stronger hydrophobic attraction, then this could explain how TMAO acts to stabilize folded protein states, hence, countering urea-induced denaturation. In fact, we find that the opposite is true. TMAO essentially removes the hydrophobic attraction, both in water-TMAO and in water-urea-TMAO solutions. The four systems considered are summarized in Table 1. In each case, the central simulation cell contains 500 particles in total, 2 of which are neopentane molecules. Note that the molarities of urea and TMAO vary, but remain roughly in the vicinity of 8 and 4 M, respectively. All molecular dynamics simulations were performed in the NVT ensemble at 298 K. For each system, the length of the cubic simulation cell was adjusted to give a pressure of ∼1 atm. The potentials of mean force were obtained by first calculating the average force acting between a pair of neopentane particles as a function of separation and then integrating. For each neopentane separation, the system was equilibrated for 400 ps, and averages were then calculated over an additional 3 ns. The total neopentane-neopentane PMF is the sum of direct and indirect contributions. For the model considered, the direct contribution is simply the neopentane-neopentane LJ interaction; the indirect part comes through solvent interactions, and it is in this contribution that hydrophobic effects are most clearly seen. The total potentials of mean force are plotted in © 2007 American Chemical Society
Letters
J. Phys. Chem. B, Vol. 111, No. 28, 2007 7933
TABLE 1 system
Nwatera
1 2b 3c 4b,c
498 398 458 358
a
NNP
Nureaa
NTMAOa
2 2 2 2
0 100 0 100
0 0 40 40
a Nwater, NNP, Nurea, and NTMAO are the number of water, neopentane, urea, and TMAO molecules in the simulation cell. b The urea concentrations are 8.4 and 8.9 M in systems 2 and 4, respectively. c The TMAO concentrations are 3.7 and 4.1 M in systems 3 and 4, respectively.
Figure 1. Neopentane-neopentane potentials of mean force in system 1 (open squares), system 2 (crosses), system 3 (open triangles), and system 4 (stars). The total potentials of mean force are shown in (a), and the direct (solid curve) and indirect contributions in (b). The dotted lines are to guide the eye.
Figure 1a, and the direct and indirect contributions are shown in Figure 1b. It is interesting to first compare our result for neopentane in pure water (system 1) with that of Lee and van der Vegt,10 obtained for the same spherical neopentane model, but using the TIP4P potential for water, rather than the SPC/E model that we employ. We note that the position of the first minimum in the total PMF (Figure 1a) at ∼6.5 Å is in good agreement with the earlier result, but that the well depth we observe (∼-5 kJ mol-1) is nearly twice that found by Lee and van der Vegt. To understand the origin of this discrepancy, we carried out a calculation for system 1 using the TIP4P water model, and the PMF we obtained (not shown) was in good agreement with that of Lee and van der Vegt. This illustrates
that in these systems, the PMF can be rather sensitive to details of the models employed, as previously noted by Lee and van der Vegt.10 As discussed above, we are interested in the influence of solutes on the PMF and, in particular, in any changes they induce in the hydrophobic attraction. Such effects are obviously best understood by considering the indirect contributions to the PMF plotted in Figure 1b. Focusing on these plots, we note that a hydrophobic attraction is obvious in the pure water (system 1) result and that this curve is not greatly modified by the addition of urea (system 2). For the models we consider, urea has only a small influence in the vicinity of the attractive well in the total PMF, but it does increase the height of the second maximum (Figure 1a). This differs somewhat from the result of Lee and van der Vegt.10 In their calculations, urea slightly weakens the hydrophobic attraction at ∼6.5 Å, but the second peak does increase in height, which is consistent with our observation. In all likelihood, the differences observed are due to the different water and possibly urea models employed. Interestingly, we also see from Figure 1b that TMAO has a much larger influence than urea, effectively negating the hydrophobic attraction in both the binary water-TMAO solution (system 3), and in the ternary water-urea-TMAO case (system 4). For these systems, the attractive “contact” interaction that remains in the total PMF comes entirely through the direct LJ contribution. The complete destabilization of the hydrophobic attraction by TMAO would lead one to argue that TMAO should favor unfolded or denatured protein states, whereas exactly the opposite is observed experimentally. This lends support to our recent suggestion6 that preferential solvation of TMAO by water and urea is a likely explanation for its tendency to counter protein denaturation in water-urea solution. Acknowledgment. The financial support of the Natural Science and Engineering Research Council of Canada is gratefully acknowledged. This research has been enabled by the use of WestGrid computing resources, which are funded in part by the Canada Foundation for Innovation, Alberta Innovation and Science, BC Advanced Education, and the participating research institutions. WestGrid equipment is provided by IBM, Hewlett-Packard, and SGI. References and Notes (1) Daggett, V. Chem. ReV. 2006, 106, 1898 and references therein. (2) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. 2003, 100, 5142. (3) Caballero-Herrera, A.; Nordstrand, K.; Berndt, K. D.; Nilsson, L. Biophys. J. 2005, 89, 842. (4) Mountain, R. D.; Thirumalai, D. J. Am. Chem. Soc. 2003, 125, 1950. (5) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. 2004, 101, 6433. (6) Paul, S.; Patey, G. N. J. Am. Chem. Soc. 2007, 129, 4476. (7) Wallqvist, A.; Covell, D. G.; Thirumalai, D. J. Am. Chem. Soc. 1998, 120, 427. (8) Ikeguchi, M.; Nakamura, S.; Shimizu, K. J. Am. Chem. Soc. 2001, 123, 677. (9) Shimizu, S.; Chan, H. S. Proteins 2002, 49, 560. (10) Lee, M.; van der Vegt, N. F. A. J. Am. Chem. Soc. 2006, 128, 4948. (11) Athawale, M. V.; Dordick, J. S.; Garde, S. Biophys. J. 2005, 89, 858. (12) Kuharski, R. A.; Rossky, P. J. J. Am. Chem. Soc. 1984, 106, 5786; 106, 5794.
