Mutation of Charged Residues to Neutral Ones Accelerates Urea

Aug 20, 2010 - Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of tw...
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Mutation of Charged Residues to Neutral Ones Accelerates Urea Denaturation of HP-35 Haiyan Wei,† Lijiang Yang,‡,§ and Yi Qin Gao*,†,‡,§ Department of Chemistry, Texas A&M UniVersity, P.O. Box 3012, College Station, Texas 77842, College of Chemistry and Molecular Engineering, Peking UniVersity, Beijing 100871, China, and Beijing National Laboratory for Molecular Sciences, Beijing 100871, China, ReceiVed: April 26, 2010; ReVised Manuscript ReceiVed: July 15, 2010

Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of R-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of β-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea. Introduction During recent years, the mechanism of urea induced protein denaturation has been widely studied by using molecular dynamics simulations.1-36 There has been considerable interest in understanding whether the direct or indirect effect or the combination of the two provides the driving force in ureainduced protein denaturation. The indirect mechanism presumes that urea denatures proteins by disrupting the water structure, which in turn weakens the hydrophobic effect and makes the hydrophobic groups more readily solvated,1,20,37-39 whereas the direct mechanism hypothesizes that urea unfolds proteins through direct hydrogen bonding with protein backbone,38 through electrostatic interactions with polar residues,2,14,16,40,41 or through van der Waals attractions with residues at protein surfaces.13,27,42,43 On the basis of earlier molecular dynamics simulations44,45 of the urea-induced denaturation of a number of β-hairpin structures, we proposed that urea induces protein denaturation through the combination mechanism of both “indirect” and “direct” interactions of urea with protein backbone: First, urea molecules accumulate around the protein backbone to displace a portion of water molecules and allow the remaining water to interact more efficiently with the protein backbone which become more competitive in forming hydrogen bonds with the protein backbone, especially the CO groups. This interaction between water and peptide backbone CO groups triggers the breaking of protein native hydrogen bonds, as an indication of an “indirect” effect of urea. The free amide group become hydrogen bonded with urea (and to a less extent, with water). The solvent/protein hydrogen bond formation prevents the * To whom correspondence should be addressed. E-mail: yiqinwfaw@ gmail.com. † Texas A&M University. ‡ Peking University. § Beijing National Laboratory for Molecular Sciences.

β-hairpin peptide from folding back to its native structure, indicating a strong direct effect of urea-induced peptide denaturation. The preferred hydrogen bonding between urea and the protein NH groups is consistent with the recent hydrogen exchange experiments.46 Further, using carbon nanotubes as a model hydrophobic system,47 it was found that urea increases the hydration of the interior pore of the carbon nanotube, although the pore was chosen to be small to avoid urea penetration. Urea molecules were found to be preferred bound to the exterior of the nanotube. The analysis of the interaction energies of the systems showed that the preferred urea binding to a hydrophobic surface does not only directly increase the interaction between the cosolvent and the hydrophobic solute, but also stabilizes the solvent inside the small and compact hydrophobic cores. These results on the model system again suggest that both direct and indirect mechanisms can be important in the kinetics of urea denaturation, and that these two types of effects are strongly related. Similar studies of Das et al.48 showed that urea preferentially binds to the carbon nanotube and dewets the latter when the pore size of the carbon nanotube is large, suggesting the preferred urea binding over water at the hydrophobic surfaces and possibly the size-dependent mechanism in urea denaturation. In the present study, we again focus on a small system and show the role of urea-induced hydration enhancement in such a system. The chicken villin headpiece (HP-35) is the smallest naturally occurring polypeptide that autonomously folds into a globular structure.49 HP-35 consists of three helices surrounding a compact hydrophobic core. The three helices are helix-1, helix2, and helix-3 for residues 4-10, 15-19, and 23-32, respectively. They are held together by a loop (residues 9 to 14), a turn (residues 19 to 22), and the phenalanine hydrophobic core of Phe6, Phe10, and Phe17. The folding time of peptide HP-35 is 4.3 ( 0.6 µs at 300 K.49 The LYS24/29NLE double mutant

10.1021/jp103770y  2010 American Chemical Society Published on Web 08/20/2010

Urea Denaturation of HP-35 of villin headpiece subdomain HP-35 NleNle is the fastest folding protein known with a folding time constant of 0.6 µs at 300 K.50 The substitution of two buried lysine residues by norleucine residues is shown experimentally to stabilize the folded structure by 1 kcal/mol and increases the folding rate by six- to seven-fold. In this paper, through a thorough analysis of the interaction of water/urea solvent with the phenalanine hydrophobic core of Phe6, Phe10, and Phe17, as well as the hydrogen bonding formation between the peptide backbone and water/urea solvent, we provide detailed molecular insights into the role of water/ urea in the expansion of the hydrophobic core as well as the breaking of the backbone native hydrogen bonds for the two polypeptides. These analyses show that there is a common mechanism in the breaking of protein backbone hydrogen bonds of both R-helix and β-hairpin structures in urea solution. And through the comparison of the denaturing process of wild peptide HP-35 and mutant peptide HP-35 NleNle, we investigate the effect of the mutant residues (LYS24/29NLE) residing on helix-3 on the stability of the polypeptides, as well as the effect on the distribution and interaction of water and urea around the hydrophobic core. Surprisingly, it was found that the mutation decreases the stability of the protein in urea solution, opposite to its effect in pure water. Simulation Methods. NPT (constant number, pressure, and temperature) ensemble calculations were performed with the AMBER 9 suite of programs and the AMBER99 force field.51 The force field for urea developed by Duffy et al. was used in the simulation.51 Periodic boundary conditions were used in the energy minimization and MD simulations. The SHAKE algorithm was used to constrain all bonds involving hydrogens.52 A cutoff of 10.0 Å was applied for nonbonding interactions. The Particle Mesh Ewald method was applied to treat long-range electrostatic interactions.53 The initial structure of wild type HP-35 was taken from its NMR structure (PDB 1YRF with the sequence of LSDEDFKAVFGMTRSAFANLPLWKQQNLKKEKGLF).54 The structure of the peptide HP-35 NleNle is taken from the PDB 2F4K.50 The terminals of both peptides are oppositely charged in the simulations. Boxes of 5 M aqueous urea solution were prepared by immersing peptide HP-35 into a cubic box containing 424 urea molecules and 3733 SPC/E55 water molecules-S1, (S2: 448 urea molecules and 3751 SPC/E55 water molecules; S3: 498 urea molecules and 4157 SPC/E55 water molecules; S4 (8M): 540 urea molecules and 3054 SPC/E55 water molecules), or immersing mutant peptide HP-35 NleNle into a cubic box containing 404 urea molecules and 3640 SPC/E water moleculesS1 (S2: 496 urea molecules and 4333 SPC/E55 water molecules; S3: 498 urea molecules and 4569 SPC/E55 water molecules; S4 (8M): 540 urea molecules and 2969 SPC/E55 water molecules). For comparison, the two peptides are also simulated in pure water: the peptide HP-35 was immersed into a cubic box containing 4094 SPC/E55 water molecules, and the mutant peptide HP-35 NleNle was immersed into a cubic box containing 4156 SPC/E water molecules. (The RMSDs and hydrogen bond survival probability are plotted in the Supporting Information and show that both polypeptides are stable in pure water, Figures S1 and S2.) The energies of the systems were minimized through a total of 2500 steps of calculations: 1000 steps of steepest descent minimization with the polypeptides being fixed by using harmonic restraints, with a force constant of 500.0 kcal mol-1 Å-2 applied to the backbone atoms, which is then followed by 1500 steps of conjugate gradient minimization. Subsequently,

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Figure 1. Time evolution of RMSDs: the backbone heavy atoms of the three helical segments with reference to the starting structure (black) and radius of gyration Rg of the phenylalanine hydrophobic core with reference to the starting structure (red) for HP-35 (Top panel) and HP35 NleNle (bottom panel).

the system was heated to 360 K and equilibrated for 1 ns at 360 K, followed by a 200 ps of cooling from 360 to 300 K performed with harmonic restraints (force constant )10.0 kcal mol-1 Å-2) applied to the backbone atoms. The production runs of 200 ns for both peptides of HP-35 and HP-35b NleNle are run and are used for the data analysis, with the data being collected every 0.5 ps. Hydrogen Bonds Definition. The number of hydrogen bonds per water or urea molecules was calculated by using the ptraj tool (inserted in the Amber package) with a cutoff radius of 3.2 Å between the hydrogen donor and acceptor and a cutoff of 135° for the O-H-O angle. Results The Denaturation of the Wild Type HP-35 and the Mutant HP-35 NleNle. Three independent simulations each with a length of 200 ns were performed for both wild type villin headpiece HP-35 and the mutant HP-35 NleNle in 5 M urea solutions. To examine the breaking of the secondary structures and the hydrophobic clustering, we show in Figure 1 the time evolution of the sum of the root-mean-square displacement (rmsd) of the three helices and the time evolution of the radius gyration (Rg) of the hydrophobic core for both peptides. The results shown in Figure 1 are obtained from the average of the three trajectories. It is clear from this figure that both wild type and mutant polypeptides gradually denature in urea solutions. One interesting difference between the two is that the denaturation onsets earlier and progresses faster for the mutant, although experiments showed that it has a shorter folding time and is more stable than the wild type. To further validate these observations, independent simulations were also performed for the denaturation of the two polypeptides in 8 M urea solutions. The calculations again show that the mutant is more easily denatured than the wild type (see the Supporting Information, Figure S3). To view some molecular details of urea denaturation, we also show in Figure 2a-d the native and denatured states of the two peptides. Since all trajectories are qualitatively similar, we use one trajectory for each polypeptide as the example to describe the denaturation process. In trajectory one, after 200 ns simulation, the denatured state of HP-35 displays a partially expanded phenylalanine hydrophobic core (formed by Phe6, Phe10, and Phe17) and a disruption of the secondary structure of helix-1, shown in Figure 2b. For HP-35 NleNle, the denaturated state is characterized by the totally expanded phenylalanine hydrophobic core and the disruption of secondary structures of helix-1 and helix-2, shown in Figure 2d. The different denaturing behavior of the wild type and mutant HP-35 in urea solutions indicates that the doubly mutant residues

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Figure 2. (a, b) The native structure and partially denatured structure for HP-35 after 200 ns of simulation; (c, d) the native structure and partially denatured structure for HP-35 NelNle after 200 ns of simulation.

Lys24Nle/Lys29Nle should play important roles in the destabilization of the mutant peptide, which is discussed later. In the following, we analyze the denaturation of the polypeptides in more detail and focus first on the expansion of hydrophobic core and then on the breaking of the R-helices. Solvent-Induced Expansion of Phenylalanine Hydrophobic Core for Both Peptides. We first describe how water and urea lead to the expansion of the hydrophobic core of the peptides. Figure 3 shows six snapshots which illustrate the solvation of the phenylalanine hydrophobic core of HP-35 NleNle. (The results for HP-35 are similar to Figure 3, and are displayed in Supporting Information, Figure S4.) In this particular trajectory, the phenylalanine hydrophobic core in HP-35 NleNle becomes solvated starting at around 54 ns. Water molecules penetrate into the phenylalanine hydrophobic core ahead of urea: In parts b anc c of Figure 3 (at around 54.15 and 54.5 ns of the simulation), several water molecules but no urea are observed residing inside the phenylalanine hydrophobic core. Urea molecules start to enter the hydrophobic core after the expansion of the hydrophobic core, induced by the penetration of water molecules. At around 54.75 ns during the simulation, Figure 3d, one urea molecule approaches the interior of the phenylalanine hydrophobic core and remains inside of the hydrophobic core for several hundred picoseconds. The same time sequence of events was observed for all individual trajectories. This observation is consistent with results on the urea denaturation of the protein chymotrypsin inhibitor 2 studied by Daggett,1 as well as the urea-enhanced hydration of the interior of carbon nanotubes.46 These studies show the “indirect effect” of urea: The addition of urea molecules into the water system changes the activity difference between water inside the hydrophobic core and in the bulk, making water molecules more active in interacting with the protein. The facilitated water penetration by urea can also be seen from the increased number of water molecules inside the hydrophobic core when urea is added (see Table 1).

Figure 3. Solvation of the hydrophobic core in mutant peptide HP-35 NleNle. Snapshots show water and urea within 4.5 Å of the side chain of the phenylalanine hydrophobic core (in space-filling representation). Water (green) is shown penetrating the phenylalanine hydrophobic core at around 54.15 ns, and urea (colored by atom) is shown to enter the hydrophobic core from around 54.75 ns after the expansion of the hydrophobic core, which is induced by water penetrating. The figure was made by using VMD.46

TABLE 1: The Average Number of Water/Urea Molecules Residing Inside the Phenylalanine Hydrophobic Core (phe6, phe10, phe17) in the Folded States of HP-35 and HP-35 NleNle in Pure Water and Urea Aqueous Solutionsa HP-35

HP-35 NleNle

pure water 5 M urea solution pure water 5 M urea solution water urea

0.0060

0.0846 0.0007

0.0126

0.1651 0.0274

a Data were collected within the first 10 ns of simulation for all four systems. (The structures of the peptide HP-35 and HP-35 NleNle remain stable during this period of time.)

To further reveal more details of the solvation of the hydrophobic core we also calculated the contact number of water/urea with the side chain aliphatic carbon of the phenylalanine hydrophobic core (Phe6, Phe10, and Phe17). The results are shown in Figure 4. In the folded states of both peptides, water and urea molecules are both in contact with the side chains of the hydrophobic core residues. The contact number of water is slightly higher than that of urea with these three residues (the first 80 ns for HP-35 and first 35 ns for HP-35 NleNle are used in the analysis of protein structures before denaturation). A significant increase is observed for the contact number of water and urea with the side chains of the hydrophobic core of both peptides in the initial denaturation process (80-135 ns for HP-35 and 35-50 ns for HP-35 NleNle). In the denatured

Urea Denaturation of HP-35

Figure 4. The contact number of water/urea molecules with the side chain of the phenylalanine hydrophobic core (phe6, phe10, phe17) for HP-35 (a) and HP-35 NleNle (b): (black) contact number of water molecules with the side chain aliphatic carbon of phe6, phe10, and phe17; (red) contact number of urea molecules with the side chain aliphatic carbons of phe6, phe10, and phe17.

state for both peptides, the average contact numbers between water (urea) and the phenylalanine residues are similar (after 135 ns for HP-35 and after 50 ns for HP-35 NleNle), both fluctuating at around ∼4-7 and indicating that the phenylalanine residues are well solvated by both solvent and cosolvent molecules except for the Phe17 in peptide HP-35. Taking into account the larger size and lower concentration of urea, these values of contact numbers show that urea is preferred over water in their interactions with the hydrophobic side chains. This quantitative analysis is consistent with earlier studies.13,32 Combining Figures 3 and 4, a physical picture appears regarding the expansion of the hydrophobic core in urea induced protein denaturation. First, urea is preferentially bound to the protein surfaces: the accumulation of urea molecules around the side chains of hydrophobic core residues of the folded protein is only slightly less than that of water molecules, despite the fact that the ratio between water/urea is around ∼8.8 in the bulk. Second, the expansion of hydrophobic core is initiated by the increase of the contact between water molecules and the side chain of hydrophobic core residues. Therefore, the accumulation of urea around the hydrophobic core residues is important in two ways: It allows the favored direct interaction of urea with protein and changes the activity of water molecules. These water molecules thus can interact more effectively with the peptides. This second and indirect effect of urea is especially important when the hydrophobic space is too small for urea to penetrate into. The penetration of water effectively induces the expansion of the hydrophobic core, which is then further solvated and stabilized through interactions with both water and urea, especially the latter. Interactions of Water/Urea with the Folded HP-35 and Mutant. As mentioned earlier, the mutation of HP-35 to HP35 NleNle increases the stability of the folded structure in water. It is thus perplexing that in urea the latter is more easily denatured. To quantitatively understand the difference in the solvation of HP-35 NleNle and the wild type HP-35 in urea solution, we analyzed the interactions between water/urea and the phenylalanine hydrophobic core of the two peptides at their native state by calculating the average numbers of water and urea molecules residing in the phenylalanine hydrophobic core. The results are given in Table 1. The interactions between water and the phenylalanine hydrophobic core residues in both peptides change significantly after the addition of urea cosolvents. For HP-35 in 5 M urea solution, the average number of water molecules residing in the phenylalanine hydrophobic core is 0.0846, which is about 14 times more than that in pure water. The average number of water molecules residing in the phenylalanine hydrophobic core

J. Phys. Chem. B, Vol. 114, No. 36, 2010 11823 is only 0.0060. This change of water population inside the hydrophobic core as a result of added urea corresponds to a change of water chemical potential difference of ∼1.6 kcal/ mol. For HP-35 NleNle, the average number of water molecules residing in the phenylalanine hydrophobic core in 5 M urea solution is 0.1651, which is about 13 times that in the pure water system. These observations indicate that the addition of urea significantly increases water interaction with the hydrophobic core of the two proteins. These results are also consistent with studies on the urea-induced hydration of the carbon nanotube interior.47 The population of water molecules residing inside the hydrophobic core for HP-35 NleNle is about twice that for HP35 (0.1651/0.0846). In addition, the probability of finding a urea inside the hydrophobic core for HP-35 NleNle is also much higher than that for HP-35. This difference between the wild type HP-35 and its mutant provides a possible explanation of the easier denaturation of the latter than the former. It is interesting to note that although water and urea penetration into the hydrophobic core is more likely for the mutant type, its hydrophobic core on the other hand appears to be more compact (e.g., as shown in Table 1, the centers of mass of its hydrophobic residues form a triangle with a smaller area compared to the wild type). It is also interesting to note that the addition of urea initially tightens the hydrophobic core but at the same time allows the solvent/cosolvent molecules to penetrate into the core more readily. It is the latter but not the former property of urea that is in line with its capability of denaturing proteins. The increased hydrophobic packing before denaturation by urea, on the other hand, is consistent with the observation that urea increases the interfacial tension between aqueous solutions and air. The different stability of the wild type and mutant HP-35 in urea solution may be a result of the NH3+ group of lysine that is in the former but not the latter. This positively charged group close to the hydrophobic core of HP-35 reduces urea accumulation at the protein surface. When the two lysines are mutated to neutral norleucine residues, the hydrophobic side chains become more closely packed. However, due to the favorable interactions between the nonpolar norleucine residues and urea the latter accumulates to a larger extent near the hydrophobic core. This accumulation of urea further brings water into the core as shown in Table 1, and thus causes the fast denaturation of the mutant peptide HP-35 NleNle. Solvent-Induced Disruption of the Secondary Structure of Helices in Both Peptides. Both water and urea molecules can serve as hydrogen donors to the backbone carbonyl groups and also as proton acceptors for the backbone amino groups in forming urea/protein hydrogen bonds. To obtain further details on the disruption of R-helices in urea-induced denaturation, we examine in the following the breaking processes of the individual native backbone hydrogen bonds for both HP-35 and its mutant. The evolution of the native backbone hydrogen bonds in helix-1 and -2 as a function of time is shown as an example in Figure 5a,b. For HP-35, the breaking of the six native backbone hydrogen bonds (HB1-HB6) in helix-1 occurs over a rather long period of time (10 to 100 ns, as seen from Figure 5a). The hydrogen bond breaking process is not of any obvious order: HB-1 first became broken at around 10 ns but reformed at around 25 ns. It is broken again at ∼50 ns. HB-2, -3, -4, and -5 are broken during the 50 to 135 ns period, which is consistent with the large increase of rmsd-R1 during this period of time, as shown in Figure S5 (Supporting Information). As another example, the breaking of backbone native hydrogen bonds in

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Figure 5. The survival probability of individual native hydrogen bonds for helix-1 (top) and helix-2 (bottom) in HP-35 (a) and HP-35 NleNle (b) in 5 M urea solution. In helix-1 (top panel): HB-1 (black), O(Ser2)fN(Phe6); HB-2 (red), O(Asp3)fN(Lys7); HB-3 (green), O(Glu4)fN(Ala8); HB-4(blue), O(Asp5)fN(Val9); HB-5 (orange), O(Phe6)fN(Phe10); HB-6(brown), O(Lys7)fN(Gly11). In helix-2 (bottom panel): HB-1 (black), O(Thr13)fN(Phe17); HB-2 (red), O(Arg14)fN(Ala18); HB-3 (green), O(Ser15)fN(Asn19); HB-4 (blue), O(Ala16)fN(lue20).

Figure 6. (a-f) The breaking and formation of hydrogen bonds for residues involved in the six backbone native hydrogen bonds in helix-1 (HB-1 to HB-6): (black) number of backbone native hydrogen bonds; (red) number of hydrogen bonds formed with water; (blue) number of hydrogen bonds formed with urea. The solid lines represent hydrogen bonds in which water or urea served as hydrogen donors and the dashed ones are for hydrogen bonds in which water or urea are proton acceptors.

helix-1 of HP-35 NleNle is shown in Figure 5b (top panel), which occurred during a short period of time between 35 and 45 ns, again without any obvious order. The breaking of R-helix hydrogen bonds in urea denaturation is in obvious contrast to that of β-hairpin hydrogen bonds. The latter shows a stronger dependence on the position of the hydrogen bonds relative to the β-turn as well as the hydrophobic core.36,44 To investigate the role of water/urea solvent participating in inducing the breaking of backbone native hydrogen bonds in proteins and to compare earlier studies on β-hairpin systems,36,44 we analyzed the hydrogen bond formation between solvent/ cosolvent and carbonyl/amino groups of the residues in helix1. The analysis was on amide groups that form backbone native hydrogen bonds 1 to 6 in wild peptide HP-35, shown in Figure 6a-f. (The hydrogen bond formation between water/urea and the carbonyl and NH groups of the residues that form native hydrogen bonds from helix-1 and helix-2 in mutant peptide HP35 NleNle is similar to that in Figure 6a-f and is not shown.) It can be seen from Figure 6a-f that during the breaking of six native hydrogen bonds HB1-HB6, the formation of a hydrogen bond between the protein CdO group and water as a proton acceptor is more likely to occur before the formation of hydrogen bond between protein CdO groups and urea. The formation of hydrogen bonds between water and the protein backbone N-H group typically follows the breaking of the

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Figure 7. (a, b) The breaking and formation of hydrogen bonds for residues involved in all the native backbone hydrogen bonds for HP35 (a) and HP-35 NleNle (b): (black) number of backbone hydrogen bonds; (red) number of hydrogen bonds formed with water; (blue) number of hydrogen bonds formed with urea. The solid lines represent hydrogen bonds in which water or urea serve as the hydrogen donors and the dashed ones are for hydrogen bonds in which water or urea are proton acceptors.

backbone native hydrogen bonds, i.e. after the formation of the backbone CdO/water (or urea) hydrogen bond. The formation of the hydrogen bond involving NH is thus more likely a consequence of breaking of backbone native hydrogen bonds and does not appear to be required for breaking of the amide hydrogen bond as indicated by the sequence of events shown in Figure 6a-f. These results suggest that the breaking of backbone native hydrogen bonds is most likely initiated by the attack of the protein amide CdO group by water and/or urea. And water is more likely to initiate the process of breaking the backbone hydrogen bonds. The penetration of water into the interior of the protein backbone that initiates the denaturation of protein was also observed in MD simulation on urea denaturation of another secondary structure of β-hairpin peptides: GB1, PEPTIDE 1, and TRPZIP4. This common mechanism of protein backbone hydrogen bond denaturation by urea solution suggests that the indirect effect of urea in promoting the water/CdO interaction plays an essential role in the kinetics of protein denaturation. The effect of urea on water/protein interaction allows the initiation of protein secondary structure breaking, and the direct interactions between urea/water and denatured protein help stabilize the denatured structures. And to evaluate the overall hydrogen bonding between water/ urea and the proteins, we show in Figure 7 the time evolution of formation probability for backbone hydrogen bonds in the helices as well as hydrogen bonds between backbone residues and urea/water for HP-35 and HP-35 NleNle, respectively. The results shown here are obtained from the average of three trajectories. As seen from Figure 7, a significant number of hydrogen bonds are already formed between backbone carbonyl and water/urea in the folded state of both peptides, whereas there are very few hydrogen bonds formed between backbone NH groups and water/urea. This observation is consistent with the notion that urea does not play an important role in serving as a proton acceptor during the disruption of the backbone hydrogen bonds. Figure 7 also shows that in the denaturing process of the breaking of backbone hydrogen bonds in helices, the breaking of the backbone hydrogen bonds is typically associated with the hydrogen bonds formation between backbone carbonyl and water/urea. However, in the denatured state of both polypeptides, a significant increase of the number of backbone NH/urea hydrogen bonds is detected when compared with the folded states, indicating that the hydrogen bonding between water/urea and protein NH groups might serve to stabilize the denatured protein. This latter observation is consistent with earlier simulations by Berne and co-workers32 as well as hydrogen exchange experiment.46

Urea Denaturation of HP-35 Therefore, we conclude that during the hydration of the peptide backbone, it is the formation of hydrogen bonds between the backbone carbonyl group and water/urea inducing the breaking of the backbone native hydrogen bonds. The formation of hydrogen bonds between the backbone amino group and urea only appears to stabilize the denatured unfolded structure, but does not play an important kinetic role during the breaking of protein backbone native hydrogen bonds, which is a strong direct effect of urea. These conclusions are consistent with a previous observation obtained from the denaturation of the β-hairpin secondary structure of peptide 1 and TRPZIP4, GB1 in urea solution. Conclusion In this study, we used all-atom MD simulations to investigate the molecular mechanism of urea induced R-helical peptide denaturation. Two R-helical bundle polypeptides, HP-35 and its mutant HP-35 NleNle that results from the substitution of the two lysine residues with norleucine residues Lys24Nle/ Lys29Nle, were used as model systems. In this study we seek answers to the following questions: (1) What is the molecular mechanism of the breaking of backbone hydrogen bonds of R-helices? Is this mechanism the same as that observed for β-hairpin structures? (2) What is the role of urea in the breaking of hydrophobic cores? Is there an indirect effect as observed in the hydration of the carbon nanotube interior? (3) What is the influence of the mutation of two charged residues on the denaturation of HP35? What is the molecular mechanism of this difference? First, it was found that similar to β-hairpins, the backbone hydrogen bonds of R-helices are broken as a result of both direct and indirect effects of urea: The preferred binding of urea to the protein surface allows adjacent water molecules to interact more favorably with the carbonyl group of the protein backbone. The hydrogen bonding between water and carbonyl groups initiates the breaking of the protein hydrogen bonds. The denatured structure is further stabilized by the hydrogen bonding of the protein backbone with both solvent and cosolvent molecules. Second, both direct and indirect effects were also observed for the breaking and solvation of the protein hydrophobic core: Even in the very early stage of simulations during which the hydrophobic packing remained compact, the addition of urea significantly increases the probability of water penetration into the hydrophobic core. This occupation of water inside the hydrophobic core, similar to the preferred hydration of the hydrophilic backbone, is also correlated to the preferred binding of urea near the protein surface and can be understood as the reduced water activity when it interacts with protein compared to that in the bulk. Furthermore, it was found that the mutant HP-35 NleNle, with two mutant norleucine residues residing on the helix-3, is more easily and quickly denatured in urea aqueous solution, whereas the wild peptide HP-35, with two lysine residues residing on the helix-3, is significantly more stable in urea aqueous solution compared to the mutant. These results suggest that the substitution of the two lysine residues with neutral non-natural residues plays an important role in facilitating the denaturation of mutant peptide. From the analysis of the distributions of solvent and cosolvent near the proteins, it was found that the NH3+ group in the two lysine residues weakens urea binding to the peptide, which consequently reduces water molecules accumulation inside the hydrophobic core. The attenuated solvation of the hydrophobic cluster thus causes the slow denaturation of the wild type HP-35. In summary, combined with the observations obtained from previous studies on a number of β-hairpins as well as on the

J. Phys. Chem. B, Vol. 114, No. 36, 2010 11825 hydration of the hydrophobic carbon nanotube, we conclude that urea denaturation of proteins involves both direct and indirect effects. Both effects are involved in the expansion of the hydrophobic core as well as in the hydrogen bond breaking in both R and β secondary structures. Urea molecules accumulate around the side chains of the protein surface and allow better hydration of the hydrophobic side chains and hydrophilic backbone. Second, urea molecules accumulate around the protein backbone to displace some of the water molecules and allow the remaining water to escape from their own hydrogen bond network and to interact more strongly with the protein backbone or the hydrophobic core. The urea-facilitated waterbackbone CO hydrogen bond formation is indicative of an indirect mechanism of urea-induced peptide denaturation. Subsequently, hydrogen bonds are formed between backbone carbonyl groups and urea molecules. Finally, the freed amide group becomes hydrogen bonded with urea (and to a much less extent, to water). The preferential hydrogen bond formation between urea oxygen atoms and peptides backbone amino groups stabilizes the unfolded conformation and prevents the peptide from folding back to its native structure, which is again indicative of a direct effect of urea-induced peptide denaturation.13 Acknowledgment. Part of the simulations were performed using the Brazos, Madusa, and Hydra supercomputers of Texas A&M University. L.Y and Y.Q.G. thank the support of Peking University. Y.Q.G. is a 2006 Searle Scholar and a 2008 Changjiang Scholar. Supporting Information Available: The simulation trajectories for HP-35 and HP-35 NleNle in pure water (Figure S1) and the hbonds survival probability of the two simulations (Figure S2), simulation trajectories for HP-35 and HP-35 NleNle in 8 M urea solution (Figure S3), solvation of the hydrophobic core in peptide HP-35 (Figure S4), and three simulation trajectories for HP-35 and HP-35 NleNle in 5 M urea solutions (Figures S5, S6, and S7). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (9), 5142–5147. (2) Bennion, B. J.; DeMarco, M. L.; Daggett, V. Biochemistry 2004, 43 (41), 12955–12963. (3) Feng, J. H.; Li, X. J.; Pei, F. K.; Chen, X.; Li, S. L.; Nie, Y. X. Anal. Biochem. 2002, 301 (1), 1–7. (4) Kresheck, G.; Scheraga, H. A. J. Phys. Chem. 1965, 69 (5), 1704– 1706. (5) Pace, C. N.; Marshall, H. F. Arch. Biochem. Biophys. 1980, 199 (1), 270–276. (6) Turner, J.; Soper, A. K.; Finney, J. L. Mol. Phys. 1990, 70 (4), 679–700. (7) Turner, J.; Soper, A. K.; Finney, J. L. Mol. Phys. 1992, 77 (3), 411–429. (8) Auton, M.; Holthauzen, L. M. F.; Bolen, D. W. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (39), 15317–15322. (9) Wang, A. J.; Bolen, D. W. Biochemistry 1997, 36 (30), 9101–9108. (10) Larsen, B. K.; Schlenk, D. Fish Physiol. Biochem. 2001, 25 (1), 19–29. (11) Niebuhr, M.; Koch, M. H. J. Biophys. J. 2005, 89 (3), 1978–1983. (12) Hayashi, Y.; Oshige, I.; Katsumoto, Y.; Omori, S.; Yasuda, A. J. Non-Cryst. Solids 2007, 353 (47-51), 4492–4496. (13) Hua, L.; Zhou, R. H.; Thirumalai, D.; Berne, B. J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (44), 16928–16933. (14) O’Brien, E. P.; Dima, R. I.; Brooks, B.; Thirumalai, D. J. Am. Chem. Soc. 2007, 129 (23), 7346–7353. (15) O’Brien, E. P.; Ziv, G.; Haran, G.; Brooks, B. R.; Thirumalai, D. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (36), 13403–13408. (16) Mountain, R. D.; Thirumalai, D. J. Am. Chem. Soc. 2003, 125 (7), 1950–1957.

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