pubs.acs.org/Langmuir © 2010 American Chemical Society
Hydrophobic Amino Acid Adsorption on Surfaces of Varying Wettability Travis G. Trudeau and Dennis K. Hore* Department of Chemistry, University of Victoria Victoria, British Columbia, V8W 3V6, Canada Received February 18, 2010. Revised Manuscript Received May 18, 2010 The adsorption behavior of the model nonpolar amino acid leucine at a solid surface of tunable wetting ability has been examined by means of molecular dynamics simulation. When leucine adsorbs at a highly hydrophobic surface it is found to orient itself with its nonpolar side chain toward the surface and its charged amine and carboxyl groups directed toward the bulk. When the surface is less hydrophobic, leucine alternates between two stable orientations, a standing orientation like that observed at nonwetting surfaces, and a laying orientation in which its side chain and charged groups are located nearly the same distance from the surface. These results are rationalized by a water-density-dependent ordering scheme in which interfacial water structure governs the adsorbed structures. We pay particular attention to how the structure of surface water is changed in the presence of adsorbed leucine in both orientations. We propose that a water density model may be applied as a general technique for understanding adsorption from solution at solid hydrophobic surfaces.
1. Introduction Many of the properties of proteins can be inferred from the sequence of their amino acid residues. For example, schemes exist for predicting membrane-spanning regions of proteins based on sequence alone.1 An attempt has recently been made to predict the adsorption affinity of proteins by statistical analysis of parameters such as hydrophobicity, although this is complicated by the necessity of considering many possible surface and solution conditions.2 A more robust method of predicting adsorption behavior from residue sequence will require isolation of singleamino acid effects from phenomena associated with larger peptide behavior (such as hindered rotation about peptide bonds). Research into amino acid adsorption has also been driven by their significance as an industrial product, due mostly to their use as food additives and as feed stock. Although many methods of synthesizing amino acids exist, all require separation techniques to obtain a desired product in significant purity.3 This has led to a significant body of literature regarding chromatography and adsorption of amino acids.4-9 The rapid and efficient separation of amino acids remains a major industrial goal. When adsorption of amino acids at polar or charged surfaces is experimentally observed, it tends to involve chemical reactions such as hydroxyl transfers which are often not reversible10,11 and in which desorption can involve fracturing of the molecule.12,13 The other major mode of adsorption on these surfaces is driven by electrostatic interactions between amino acids and surface sites *Author to whom correspondence should be addressed. E-mail:
[email protected]. Telephone: 250-721-7168. Fax: 250-721-7147..
(1) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105–132. (2) Vasina, E. N.; Paszek, E.; Dan V. Nicolau, J.; Nicolau, D. V. Lab Chip 2009, 9, 891–900. (3) Kusumoto, I. J. Nutr. 2001, 131, 2552S–2555S. (4) Gregorczyk, D. S.; Carta, G. Chem. Sci. Eng. 1995, 51, 807–818. (5) Krohn, J. E.; Tsapatsis, M. Langmuir 2005, 21, 8743–8750. (6) Titus, E.; Kalkar, A. K.; Gaikar, V. G. Colloid Surf. A 2003, 223, 55–61. (7) Vlasova, N. N.; Golovkova, L. P. Colloid J. 2004, 66, 657–662. (8) Basiuk, V. A.; Gromovoy, T. Y. Coll. Surf. A 1996, 118, 127–140. (9) Krohn, J. E.; Tsapatsis, M. Langmuir 2006, 22, 9350–9356. (10) Langel, W.; Menken, L. Surf. Sci. 2003, 538, 1–9. (11) Schmidt, M.; Steinemann, S. G. Fresnius J. Anal. Chem. 1991, 341, 412–415. (12) Paszti, Z.; Keszthelyi, T.; Hakkel, O.; Guczi, L. J. Phys.: Condens. Matter 2008, 20, 224014. (13) Luo, X.; Quian, G.; Sagui, C.; Roland, C. J. Phys. Chem. C 2008, 112, 2640– 2648.
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with high charge density.7,14,15 The former mode of adsorption is unacceptable for applications such as chromatography which depend on reversible and rapid adsorption and desorption. The latter mode is useful to separate charged amino acids, but not applicable to others. In addition, because the reactivity of these surfaces depends on specific interactions between surface sites and amino acid functional groups, their behavior is often dependent on factors such as the density of relevant groups at the surface,9 the extent of surface hydroxylation,10 and conditions in the aqueous phase such as pH11,16 and ionic strength.7,15 The ability to modify these factors may be useful for tuning properties of adsorption, but the need to control them creates an obstacle for industrial applications. Reversible adsorption of amino acids is thus a topic of considerable importance. The consensus is that reversible adsorption at a nonpolar surface is largely driven by hydrophobic attraction between the substrate and the amino acid side chain.4,6,15,17 The role of water as solvent is crucial in physisorption of amino acids; while polar and hydrophilic surfaces can reversibly adsorb all amino acids in vacuo,13 they have difficulty adsorbing them in aqueous environments.8,10,12 This implies that the role of water is crucial in promoting or preventing adsorption of individual residues and, by extension, in determining the adsorbed structure of larger peptides. Our simulations examine the adsorption of the nonpolar amino acid leucine at various azimuthally isotropic nonpolar surfaces. We will first describe the setup of our system, including a description of the surfaces of varying hydrophobicity. We then discuss our sampling criteria, and the structural results we obtain for the amino acid and the interfacial water molecules. Finally, we rationalize our results by applying the model of water-densitydependent ordering that we previously introduced18 to explain features of the orientation of water molecules at the interface between water and a hydrophobic surface. (14) Benetoli, L. O. B.; de Souza, C. M. D.; da Silva, K. L.; I., G. S., Jr.; de Santana, H.; A., P., Jr.; da Costa, A. C. S.; Zaia, C. T. B. V.; Zaia, D. A. M. Orig. Life. Evol. Biosph. 2007, 37, 479–493. (15) Deng, F.; Sun, Y.; Gao, Q.; Xu, W.; Xu, Y.; Wu, D.; Shen, W. J. Phys. Chem. B 2008, 112, 2261–2267. (16) Vinu, A.; Hossain, K. Z.; Kumar, G. S.; Ariga, K. Carbon 2005, 44, 530–536. (17) Gregorczyk, D. S.; Carta, G. Chem. Eng. Sci. 1995, 51, 819–826. (18) Trudeau, T. G.; Jena, K. C.; Hore, D. K. J. Phys. Chem. C 2009, 113, 20002–20008.
Published on Web 05/27/2010
DOI: 10.1021/la100716z
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Table 1. Values of ε Chosen for the Surfaces Used in the Study, with Associated Water Contact Angles and Surface Water Cutoff Distances ε/kJ 3 mol-1
contact angle/deg
z cutoff/A˚
0.550 1.10 1.93 2.75 4.13 5.50
156 151 134 125 101 84
3.0 3.0 3.5 3.8 3.9 4.0
2. Simulation Details Simulations were performed with the GROMACS package.19 Systems consisted of 3840 SPC/E water molecules20 and a single zwitterionic leucine molecule together with a pair of surfaces described below. The simulation cell had initial dimensions of 42 42 70 A˚3. van der Waals forces were cutoff at a radius of 9 A˚, while electrostatic interactions were handled using the PME method with a real-space cutoff of 9 A˚. Steele 10-4 potential21,22 surfaces were placed in the simulation cell at z = 0 A˚ and z = 70 A˚. This provides Lennard-Jones interactions integrated over the surface according to " 4 # 10 2 σ σ UðzÞ ¼ 2πσ2 ε 5 z z
ð1Þ
where σ is the distance along the z-axis from the surface at which the potential will be zero and ε is the depth of the potential well near the surface. In this study, σ was kept at a constant value of 3 A˚ and the values of ε were varied to produce surfaces of varying wetting ability. The surfaces used had corresponding contact angles shown in Table 1, calculated in a previous paper18 by a recently described method.23 Leucine intramolecular interactions and leucine-water interactions were handled using the OPLS-AA/L force field.24 Harmonic terms in the potential energy function arising from dihedral positions within the protein were removed to allow more conformational flexibility. Bond rotation in the amino acid was thus governed implicitly by other interactions (for example van der Waals interactions between 1-4 atoms). As a result, specially parametrized 1-4 interactions within the amino acid specified by the OPLS-AA/L force field were also excluded, and these atom pairs interacted as if nonbonded. The energy of each system was minimized three times, using the steepest-descent method implemented in GROMACS with step sizes of 0.01 A˚, then 0.05 A˚, and finally 0.1 A˚. This stepwise approach to energy minimization helped move the system out of unstable starting configurations. Following minimization, to maximize computational efficiency, nine parallel simulations of each system were run simultaneously on 8 processors each. Each simulation was equilibrated separately for 200 ps with different random velocities assigned to each of its atoms at start-up to achieve an overall temperature of 300 K. Afterward, each was run for 10 ns with data collection every 50 fs. The 10 ns runs were combined to produce the equivalent of a 90 ns trajectory of each system for analysis. The integrator step size during equilibrations (19) van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. J. Comput. Chem. 2005, 26, 1701–1718. (20) Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. J. Phys. Chem. 1987, 91, 6269–6271. (21) Steele, W. A. J. Phys. Chem. 1978, 82, 817–821. (22) Stecki, J. Langmuir 1997, 13, 597–598. (23) Hirvi, J. T.; Pakkanen, T. A. J. Chem. Phys. 2006, 125, 144712. (24) Kaminski, G. A.; Friesner, R. A. J. Phys. Chem. B 2001, 105, 6474–6487.
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Figure 1. The leucine molecule, with the positions of the center of geometry PCOG, zwitterionic midpoint P(, and average methyl position PCH3 labeled. The long axis VL is defined as the vector that points from PCH3 to P(.
and runs was 1 fs. Temperature was maintained at 300 K using a Berendsen thermostat25 throughout equilibrations and runs. Pressure was maintained at 1.01325 bar with a Berendsen barostat25 during equilibration only by varying the x and y cell dimensions while keeping the z dimension (normal to the surface) constant. Pressure coupling changed the dimensions of the simulation cell by only a small amount (approximately 1% along the x and y dimensions during equilibration), and the size of the cell had reached an equilibrium value within the first 10 ps, validating the decision to remove pressure coupling during the run. To render the two interfaces in the simulation equivalent during analysis, the coordinates of each atom above the midpoint of the box on the z-axis were rotated 180 around the y-axis.
3. Data Analysis and Results All analyses were conducted using several reference points and a vector, illustrated in Figure 1. The leucine molecule’s center of geometry, PCOG, was located by averaging the location of every atom in the molecule. The center of geometry was chosen as a reference point for analysis because it provided a measurement of leucine’s position relatively independent of its conformation. P( was defined as the average of the position of the nitrogen of the amine substituent and the carbon of the carboxy substituent. It was intended to provide a measurement of the location of the most hydrophilic, charged portion of the leucine molecule. PCH3 was defined as the average of the positions of the two carbons of the methyl groups that terminate leucine’s side chain. It was chosen to provide a marker for the most hydrophobic, nonpolar region of the leucine molecule. The vector running from PCH3 to P(, called VL, was used as a marker of leucine’s long axis orientation with respect to the surface normal. This vector runs along the longest axis of the molecule without being especially sensitive to conformation. Leucine Adsorption Trajectories. The initial analyses involved sampling the positions of PCOG, P(, and PCH3 and the orientation of VL with respect to the surface normal. Since the surfaces were isotropic in the x and y dimensions, position was recorded along the z axis only. Figure 2 shows one representative 10 ns trajectory for the 134 water contact angle (CA) and 84 CA surfaces. This analysis provided a picture of how the systems evolved dynamically, and what behaviors were associated with one another. A qualitative inspection of Figure 2 shows that events such as shifts in orientation and leucine’s adsorption and desorption from the surface are apparent, and many of these events were found to occur within the period of the simulation. The trajectories confirmed that leucine was leaving the surface for some period in each simulation and was tumbling freely during (25) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Dinola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684–3690.
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Figure 2. Examples of raw data from two 10 ns trajectories for (a, b) 134 CA surface and (c, d) 84 CA surface. In each case, the upper panels (a,c) show the positions along the z-axis of P( (blue), PCOG (red), and PCH3 (green) for leucine corresponding to the definitions in Figure 1. Leucine was considered adsorbed on the surface (yellow shaded regions) when PCOG < 7.5 A˚; this limit is shown as the dashed line (a, c). The lower panels (b, d) show the angle of the long axis vector VL relative to the surface normal. Leucine was considered laying on the surface when 70 < θL < 110 (gray shaded regions); these limits are indicated by the set of horizontal dashed lines (b, d).
this time. This was important because it indicated that leucine was able to sample the full range of orientations it had access to in the bulk, and was not excessively prejudiced toward the orientation it assumed upon initial adsorption. On the basis of the results of all such trajectories, a definition using PCOG as a criterion was formulated to determine when leucine was considered to be on the surface. If the molecule’s center of geometry had a z-coordinate within 7.5 A˚ of either surface, it was considered adsorbed. This is indicated by the yellow shaded time periods in Figure 2. When leucine was on the surface, the relative positions P( > PCH3 were consistent throughout every trajectory. Usually, the difference in z position between the points was about 4 A˚, but there were periods, mostly in trajectories of the most-wetting systems (contact angles of 84 and 101), in which the position of the center of charge would move toward the surface by 1.5-2.0 A˚, and the average methyl position would move in the same direction approximately 0.5 A˚, with the result that the molecule appeared to be laying flat on the surface. This change in orientation could be seen most clearly by examining the angle between VL and the surface normal, θL plotted in Figure 2, parts b and d. This angle typically remained less than 60 when the molecule was adsorbed, but when P( approached closer than 7 A˚ to the surface, VL jumped to above 70, supporting a conclusion that during these periods leucine was temporarily laying flat against the surface. Langmuir 2010, 26(13), 11095–11102
For simplicity, leucine was considered to be laying when the angle between VL and the surface normal was 70 < θL < 110, indicated by the dashed horizontal lines in plots Figure 2, parts b and d. The corresponding time periods are highlighted with gray shading in Figure 2. The most nonwetting surfaces exhibited a striking feature: a tendency to order the leucine molecule perpendicular to the surface, which became stronger as the surface became more nonwetting, as is shown in Figure 3. Evidence from plots c and d in Figure 2 suggests that the standing and laying orientations are somewhat stable, as an adsorbed molecule can remain in either state for periods of more than 100 ps for the 84 and 101 CA surfaces. This would not be expected if leucine continually sampled the range of available orientations. Frames in which standingto-laying and laying-to-standing transitions occurred were used to build two videos showing leucine undergoing these transitions, without visualizing the surrounding water molecules. These are available in the Supporting Information. Atomic Density Profiles. To determine the orientation of adsorbed leucine more precisely and compare the properties of the two orientation states, probability density profiles were constructed for PCOG, P(, and PCH3. Separate profiles were created for the standing and laying orientations of leucine, which were distinguished using the angular criterion discussed above. Each DOI: 10.1021/la100716z
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Figure 5. Histogram showing orientation angles adopted by leucine’s long axis vector VL with respect to the surface normal.
Figure 3. Fraction of frames along each trajectory for which leucine is adsorbed in the laying orientation plotted against water contact angle. The time spent in the laying orientation decreases as the surface becomes more hydrophobic.
determined by counting only water molecules within 6.0 A˚ of any one of leucine’s atoms. Standing orientations are distinguished with solid lines, and laying orientations with dashed lines; PCOG is red, P( is blue, and PCH3 is green. The probability density profile of pure water at the same surface is the solid black line, while the density profile of water in the presence of leucine is the dashed black line. All density profiles have been normalized so that the area under each curve is unity. Across all systems and both orientations, PCH3 is closer to the surface than PCOG, and P( is further from the surface than PCOG. In the standing orientation, the separation between PCH3 and P( is greater than in the laying orientation. In the laying orientation, P( moves considerably closer to the surface, while PCH3 moves somewhat further away from it. Leucine Orientation. A histogram was constructed to quantitatively examine leucine’s orientation at the surface, shown in Figure 5. Every 50 fs of the simulation, if PCOG was within 7.5 A˚ of either surface, a snapshot was taken for analysis. The angle between VL and the nearest surface normal was binned to 1 resolution. Each bin was divided by sin θL to normalize with respect to an isotropic orientation distribution. The results of a bulk trajectory analysis are also included in Figure 5 for comparison. When combined with the results of the trajectory analysis, Figure 5 suggests the existence of at least two distinct orientational states for leucine on the surfaces studied. These are the socalled standing and laying states that we have identified in the adsorption trajectories. To further characterize the structure of leucine, we have calculated order parameters for θL in the standing and laying orientations. The tilt order parameter, ÆPθæL, is the second term of the Legendre polynomial series. ÆPθ æL ¼
plot in Figure 4 shows the standing and laying probability density profiles associated with a particular surface, together with the density profile of pure water for the same system, and the density profile of water in the presence of leucine. The latter was 11098 DOI: 10.1021/la100716z
ð2Þ
where the angular brackets denote an ensemble average. Results of this determination appear in two columns of Table 2. A value of ÆPθæL = 0 indicates an isotropic distribution of tilt angles. If all molecules are perfectly aligned with θL = 0 (ideal standing, VL ( z) then ÆPθæL = 1. If all molecules are perfectly aligned with θL = 90 (ideal laying, VL ^ z) then ÆPθæL = -0.5. The results indicate that leucine in its standing orientation is most ordered at the most hydrophobic surface (ÆPθæL = 0.53 when the water CA is 156) and the degree of ordering gradually decreases as the surface becomes more wetting. Likewise, when leucine is adsorbed in its laying orientation, it is most ordered at the 84 CA surface with ÆPθæL = -0.43. Interfacial Water Orientation. In addition to studying the density of water before and after leucine adsorption, we have studied the change in orientation of the first layer of water )
Figure 4. Density profiles for the position of leucine’s center of geometry (red), center of charge (blue), and average methyl position (green) when adsorbed on a surface with water contact angle (a) 156, (b) 151, (c) 134, (d) 125, (e) 101, and (f) 84. Solid colored lines depict positions when leucine is in the standing orientation, dotted colored lines show positions when the molecule is in the laying orientation. The solid black line shows the probability density of water molecules at each surface in the absence of leucine (before adsorption); the dotted black line shows the probability density of water molecules in the vicinity of adsorbed leucine, defined as being within 6 A˚ of any atom of the adsorbed leucine molecule.
1 Æ3 cos2 θL - 1æ 2
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Table 2. Tilt Order Parameters for Leucine and Tilt/Twist Order Parameters for Interfacial Water Molecules without Leu
Leu standing
Leu laying
water CA/deg ÆPθæW ÆPψæW ÆPθæW ÆPψæW ÆPθæL ÆPθæW ÆPψæW ÆPθæL 156 151 134 125 101 84
-0.22 -0.22 -0.26 -0.23 -0.24 -0.24
0.13 0.10 -0.12 -0.24 -0.31 -0.35
-0.22 -0.22 -0.22 -0.22 -0.23 -0.23
0.18 0.15 -0.06 -0.18 -0.26 -0.31
0.53 0.53 0.57 0.39 0.32 0.30
-0.22 -0.24 -0.23 -0.23 -0.24 -0.25
0.15 0.07 -0.13 -0.24 -0.32 -0.36
-0.40 -0.40 -0.42 -0.40 -0.38 -0.43
molecules when leucine is adsorbed in the standing and laying orientations. In this analysis we calculate the water tilt θW as the angle between the molecular C2 symmetry axis and surface normal; the twist ψW is defined as the angle between the plane of the water molecule and a plane perpendicular to the surface. (A water molecule lying in the plane of the surface would have θW = 90 and ψW = 90.) Leucine was considered to be adsorbed and in the standing or laying orientation using the same criteria as described above. In frames where leucine was determined to be adsorbed, a water molecule was considered to be in the first layer (between the surface and the amino acid) if the z-component of its oxygen atom was less than a cutoff distance, on average 3.5 A˚. This cutoff was adjusted based on distinction between first and second layer water orientation as determined from the neat solid-liquid interfaces;18 the resulting values appear in the last column of Table 1. Figure 6 displays the joint tilt-twist histograms for the first layer of water molecules far from leucine (column 1), under standing leucine (column 2), and under laying leucine (column 3). The rows are arranged in decreasing hydrophobicity of the surface, with the first row corresponding to 156 water CA, and the last row 84 water CA. In each subplot, ψW is along the vertical axis and θW along the horizontal axis. Brighter areas (red) indicate larger populations of water; darker areas (blue) indicate lower populations. The contrast has been enhanced by median filtering with a kernel size of 7 pixels; this has been particularly useful for the visualization of low populations. At the two most hydrophobic surfaces (156 and 151 CA) the orientation of surface water molecules appears to be the same regardless of whether leucine is adsorbed. It is difficult to comment on the fine details of plots M and N due to the low population of water under leucine in the laying orientation. Once we reach a CA of 134, we observe that the population of water molecules with θW = 50, ψW = 0,180 is greater when leucine is adsorbed standing (I) than for the neat surface (C). This trend continues for the 125 and 101 CA surfaces. For the 101 and 84 CA surfaces, a population with θW = 130, θW = 0,180 develops at the neat interface (E,F) and is significantly enhanced when leucine is adsorbed in a standing orientation (K, L). We define the water tilt and twist order parameters as ÆPθ æW ¼
1 Æ3 cos2 θW - 1æ 2
ð3Þ
ÆPψ æW ¼
Æsin2 θW cos 2ψW æ Æsin2 θW æ
ð4Þ
where the limits -0.5 < ÆPθæW < 1 are defined in a manner analogous to that of the leucine tilt axis order parameter in eq 2. The twist order parameter is defined in the range -1 < ÆPψæW < 1, where ÆPψæW = -1 indicates all water molecules are twisted to lie in the plane of the interface, ÆPψæW = 1 has all water molecules Langmuir 2010, 26(13), 11095–11102
Figure 6. Histograms showing the twist ψW versus tilt θW angle of water molecules in the first layer next to the solid surface. Hydrophobicity of the surfaces decreases from CA 156 (top row) to CA 84 (bottom row). The first column (A-F) represents water far from leucine; the middle column (G-L) represents water under leucine adsorbed in the standing orientation; last column (M-R) represents water under leucine adsorbed in the laying orientation.
perpendicular to the plane of the interface, and ÆPψæW = 0 indicates no preference for the orientation of the water twist axis. Values of these two order parameters were determined for water molecules in the first layer next to the solid surface in the case where leucine is far away, leucine is adsorbed in a standing orientation, and leucine is adsorbed in a laying orientation. The results, summarized in Table 2, indicate that the hydrophobicity of the surface has little effect on the tilt order parameter of water molecules under Leu in either standing or laying orientations. We note that the tilt angle order does also not differ much for water molecules in the first layer in the absence of leucine; the primary differences occur in the twist order parameter. Looking at the results in the absence of leucine as a baseline, the change in ÆPψæW from positive to negative as the contact angle drops below ≈140 represents the switch from an out-of-plane orientation (ψ = 0, DOI: 10.1021/la100716z
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Figure 7. Density profiles of water at two model interfaces, corresponding to water contact angles of 84 (solid line) and 156 (broken line).
180) to an in-plane orientation (ψ = 90, 270) of the water molecules. It is interesting to note that, in the case of water between the surface and standing leucine, the twist orientation is more ordered for the most hydrophobic surfaces and less ordered for the least hydrophobic surfaces, in comparison with water at the neat solid-liquid interface. For molecules between the surface and laying leucine the ordering about the water twist axis is about the same (slightly enhanced) in comparison with the neat interface.
4. Discussion Water Density-Dependent Ordering as a Predictor of Interfacial Amino Acid Structure. In a previous paper,18 we proposed a model of density-dependent ordering to explain the structure of water at a hydrophobic surface. According to our model, the water density profile is considered as a predictor of the water structure across the interfacial region. We then hypothesized that subsequent adsorption of other molecules at hydrophobic surfaces may be understood on the basis of this water density. We now discuss this hypothesis in light of our observations of the adsorbed leucine behavior. Density profiles of a model nonwetting (156 CA) and semiwetting (84 CA) system are shown in Figure 7. At strongly nonwetting surfaces, the density profile of water will exhibit a nearly sigmoidal decay from bulk to zero density as one approaches the interface. As the contact angle decreases, a region of increased water density comes into existence close to the surface, along with regions of alternating low-and-high density toward the bulk water phase. These two systems represent the upper and lower limits of hydrophobicity in our study, and will be used to discuss leucine adsorption. Across all simulations, the most prominent tendency observed was for leucine’s side chain, and in particular the methyl groups that terminate it, to associate strongly with the surface. When considering the nonwetting surfaces, there is some question as to why leucine adsorbs there at all. The attractive potential exhibited by the highest contact angle surfaces is too weak to hold water molecules close to the surface; the declining water density in the vicinity of these surfaces drives leucine’s adsorption by allowing it to place its nonpolar components in regions of low water density, where they will disrupt fewer water-water interactions than in the bulk, an interfacial version of the classic hydrophobic effect. However, it is not beneficial for leucine to have its charged groups in the low-density region, since these would benefit from solvation. The solution to this dilemma is for the molecule to adopt an orientation that allows it to stretch across the low-density interface and into the bulk-density zone. The least-wetting surfaces in these simulations allow the closest approach of water molecules to the surface, since they have the shallowest repulsive potential near the surface (Figure 8). This causes the interface to be somewhat wider at the least-wetting surfaces, since smaller angular deviations will cause the amine and 11100 DOI: 10.1021/la100716z
Figure 8. Distance from the surface where greater than 0.1% of the bulk water density is observed, plotted as a function of water contact angle.
carboxyl groups to move into the low-density part of the interface. There is no benefit for leucine to orient itself with its long axis VL parallel to the surface in the least-wetting systems and, as can be seen in Figure 3, it adopts such an orientation only about 1% of the time. When fluctuations do cause it to orient itself roughly parallel to the surface, Figure 4 shows that although the methyl groups may enter the region of bulk density, the charged groups are extremely unlikely to be drawn into the interfacial region of reduced density, suggesting that solvation is the most-important consideration in determining adsorption orientation at these surfaces. The water environment near the wetting surfaces with water CA approaching or exceeding 90 is significantly different and more complicated than the nonwetting case, and leads to leucine adsorption behavior which is also more complex. In the systems with lower water contact angles, the balance between the standing and laying states is controlled by two factors: hydrophobic interactions and solvation. From a free energy perspective, it is advantageous for leucine to adsorb at the least hydrophobic surfaces by placing its hydrophobic side-chain in a region of low water density where it will have little effect on water structure. This force stabilizes the core of the leucine molecule, including most of its side chain, which is centered in the region of low water density adjacent to the initial high-density region close to the surface. Conversely, this force destabilizes the position of the methyl groups, particularly in the standing orientation. The methyl groups appear in a region of very high water density close to the surface, where they are likely to incur a significant free energy penalty from the hydrophobic effect. Adsorption is also influenced by the need to solvate leucine’s charged amine and carboxyl groups. Solvation effects stabilize the positions of the amine and carboxy groups in the standing orientation, and destabilize them in the laying orientation. In the standing orientation, both charged groups are projected into a region of bulk or greater-than-bulk water density adjacent to the low-density region where the body of leucine rests. In the laying orientation, however, the charged groups are forced into the low-density region where solvation may become more difficult. For the 84 CA surface, leucine spends about 20% of its time in the laying orientation, and for the 101 CA surface it spends about 10% of its time in this orientation, suggesting that solvation of the charged groups is the dominant effect in determining orientation, as it was for the nonwetting surfaces. The behavior of water in the presence and absence of leucine supports these conclusions. When leucine is present at the interface, there is a relative decrease in the number of water molecules very close to the surface, owing to hydrophobic repulsion with the side chain. There is a relative Langmuir 2010, 26(13), 11095–11102
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increase in water density further from the surface owing to solvation of the carboxyl and amine groups, which are directed away from the surface in the dominant standing orientation. Switching between these two orientations disrupts the water molecules involved in solvation, and the associated energy penalty provides each orientation with a degree of kinetic stability. Interfacial Water Structure. From the density profiles (Figure 4), we have observed that the interfacial water structure is not drastically altered in the presence of the hydrophobic amino acid. This is confirmed in the similarity of the gross features in the orientation histograms (Figure 6) before and after leucine adsorption. However, the subtle features of interfacial water structure reveal some intriguing trends which are illustrative of the difference in water ordering near an extended hydrophobic surface and a relatively small hydrophobic molecule. There has been considerable interest in water confined between two hydrophobic surfaces,26-30 in nanopores,31-34 and inside nanotubes.29,35 The degree of confinement in most of these studies is not as high as is experienced by water in the current study, with the minimum surface separation being about 10 A˚. However, it is generally understood that, while extended surfaces encourage water molecules to point one hydrogen-bonding group toward them, hydrophobic molecules of sufficiently small diameter encourage the formation of short-lived clathrate-like water arrangements.36,37 These structures allow water to solvate small hydrophobic moieties without sacrificing hydrogen bonding opportunities. In the current study, the effects of this transient cage-type structuring around leucine’s side chain are visible in Figure 6 plots G-L as an enhancement of water molecules with an out-of-plane orientation. This effect can be quantified through our defined order parameters. Leucine in the standing orientation is found to increase the twist order parameter by approximately 0.05 across all surfaces, supporting ordering of water out-of-plane and discouraging ordering of water within the plane of the interface. Interestingly, leucine in the laying orientation does not have an appreciable effect on water ordering at the interface. It may be that this orientation presents too large a surface area to interfacial water to allow the formation of a clathrate-like structure around it, and the influence of the surface on water structure predominates. Comparison with Experimental Studies of Amino Acid Adsorption. Experiments have suggested that, in some cases, nonpolar amino acids actually adsorb more strongly to hydrophilic than hydrophobic surfaces.4,38 This finding indicates that something more than the hydrophobic effect is responsible for adsorption, and has led to some conflicting claims in the literature. One study38 found that the free energy of adsorption of leucine was higher on a hydrophilic than a hydrophobic surface. The authors suggested the greater free energy of adsorption was an enthalpic effect arising from electrostatic interactions between (26) Urbic, T.; Vlachy, V.; Dill, K. A. J. Phys. Chem. B 2006, 110, 4963–4970. (27) Jensen, M.; Mouitsen, O. G.; Peters, G. J. Chem. Phys. 2004, 120, 9729– 9744. (28) Kumar, P.; Buldyrev, S.; Starr, F.; Giovambattista, N.; Stanley, H. E. Phys. Rev. E 2005, 72, 051503. (29) Cicero, G.; Grossman, J.; Schwegler, E.; Gygi, F.; Galli, G. J. Am. Chem. Soc. 2008, 130, 1871–1878. (30) Gordillo, M. C.; Nagy, G.; Marti, J. J. Chem. Phys. 2005, 123, 054707. (31) Liu, Y.-C.; Wang, Q.; Lu, L.-H. Chem. Phys. Lett. 2003, 381, 210–215. (32) Zhang, Q.; Chan, K.-Y.; Quirke, N. Mol. Simul. 2009, 35, 1215–1223. (33) Floquet, N.; Coulomb, J. P.; Dufau, N.; Andre, G. J. Phys. Chem. B 2004, 108, 13107–13115. (34) Kocherbitov, V. J. Chem. C 2008, 112, 16893–16897. (35) Thomas, J. A.; McGaughey, A. J. H. J. Chem. Phys. 2008, 128, 084715. (36) Lee, C. Y.; McCammon, J. A.; Rossky, P. J. J. Chem. Phys. 1984, 80, 4448– 4455. (37) Chandler, D. Nature 2005, 437, 640–647. (38) Aladdine, S.; Nygren, H. Coll. Surf. B 1995, 6, 71–79.
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leucine and a hydrophilic surface. Another work4 found that the free energy of adsorption for phenylalanine was higher on more hydrophilic surfaces as well, but they also determined that the heat of adsorption was lower for these surfaces. They concluded that entropic effects were responsible for phenylalanine’s greater affinity for hydrophilic surfaces. According to this model, the increased tendency for adsorption at a hydrophilic interface arises from the disruption and displacement of relatively ordered water layers near the surface. A separate study8 that examined the adsorption of a variety of amino acids on a model hydrophobic and hydrophilic surface, disputed a significant contribution from entropy to adsorption, and posited that it is an enthalpic phenomenon. On the basis of our findings and the density-dependent model of ordering, we propose an alternate hypothesis for the stronger affinity exhibited by nonpolar amino acids for certain hydrophilic surfaces. We propose that the preference of leucine and other nonpolar amino acids for these surfaces is in fact a consequence of the hydrophobic effect, as nonpolar regions of the amino acid are placed in regions of low water density near, but not directly adjacent to, the surface. Given that the free energy loss associated with the hydrophobic effect is mostly entropic at the temperatures in this study, we conclude that the greater affinity of nonpolar amino acids for hydrophilic surfaces is likely due to entropic effects.
5. Summary and Outlook The idea of density-dependent ordering of interfacial water at neat solid-aqueous interfaces has been applied to explain features of leucine adsorbed at a variety of solid hydrophobic surfaces. In particular, the observation that leucine switches between two distinct orientations, standing and laying, has been examined. This phenomenon has been attributed to competition between two factors: the repulsion of leucine’s side-chain from the region of high water density immediately adjacent to the surface, and the need of leucine to solvate its charged carboxy and amine groups in water of bulk-or-greater density further from the surface. Adsorption at both nonwetting and semiwetting surfaces may be rationalized in terms of the hydrophobic effect, as leucine’s nonpolar side chain is placed in a region of low water density immediately adjacent to the surface (nonwetting), or between two interfacial regions of elevated water density (semiwetting). We suggest that density-dependent ordering, the use of underlying patterns of water density as a guide to interpreting adsorption phenomena, can be of general use in explaining the adsorption of solutes at solid surfaces where hydrophobic attraction is the main factor driving adsorption. Proteins adsorbed to hydrophobic surfaces can exhibit significantly different conformation than in the bulk.39 Unlike bulk studies of proteins (by magnetic resonance, X-ray, and neutron diffraction), there are very few experimental techniques that are structurally sensitive and also selective for molecules adsorbed on surfaces. As a result, much computational effort is devoted to this area. A long-term goal of projects such as this one is to build a parameter-based model for predicting the structure of adsorbed peptides and proteins based on the sequence of residues. The first step is to fully understand the adsorption of single amino acids, and parameters such as water structure near the surface. Results from these studies could then used to refine models incorporating the effects of specific residue-residue interactions. (39) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233–244.
DOI: 10.1021/la100716z
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Acknowledgment. We wish to thank the Natural Science and Engineering Research Council of Canada (NSERC) for support of this science with a Discovery Grant. Computers were purchased with start-up funds from the University of Victoria. Supporting Information Available: Video file sl.mpg is a 50-ps clip of the transition from standing to laying (40) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33–38.
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orientation of an adsorbed leucine molecule. For clarity, water molecules have been omitted and the location of the Steele 10-4 potential barrier is illustrated by the plane of green spheres. Video file ls.mpg is a 40-ps clip of the transition from laying to standing orientation. These files were created directly from the GROMACS trajectory using the VMD package. 40 This material is available free of charge via the Internet at http:// pubs.acs.org.
Langmuir 2010, 26(13), 11095–11102
