Helix Forming Tendency of Valine Substituted Poly-Alanine: A

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Helix Forming Tendency of Valine Substituted Poly-Alanine: A Molecular Dynamics Investigation S. Sundar Raman, R. Vijayaraj, R. Parthasarathi, and V. Subramanian* Chemical Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020 India ReceiVed: December 21, 2007; ReVised Manuscript ReceiVed: April 25, 2008

In this study, classical molecular dynamics simulations have been carried out on the valine (guest) substituted poly alanine (host) using the host-guest peptide approach to understand the role of valine in the formation and stabilization of helix. Valine has been substituted in the host peptide starting from N terminal to C terminal. Various structural parameters have been obtained from the molecular dynamics simulation to understand the tolerance of helical motif to valine. Depending on the position of valine in the host peptide, it stabilizes (or destabilizes) the formation of the helical structure. The substitution of valine in the poly alanine at some positions has no effect on the helix formation (deformation). It is interesting to observe the coexistence of 310 and R-helix in the peptides due to the dynamical nature of the hydrogen bonding interaction and sterical interactions. Introduction Substitution of different amino acids in the short poly alanine (PA), based peptides has provided a perfect framework to study the helix/coil transition.1–10 A great deal of research work has been carried out on this topic due to its implication in de nova design of proteins and protein folding.11–17 These studies have helped to understand the helix formation and various physical factors governing its stability.1,2,18–21 It is evident from the results that each amino acid has a characteristic intrinsic helical propensity to stabilize R-helix.22 The hydrogen bonding interaction between i and i + 3 or i and i + 4 residues, the interaction of charged (or polar) amino acid residues with helix macrodipole, capping interaction of residues flanking the helix and the free N-H and CdO of first and last helical turns are some of important interactions leading to the stability of 310 and R helices.1,23,24 Solvent environment plays a crucial role on the helix forming tendency in addition to these interactions.25–30 Numerous molecular dynamics studies have been devoted to understanding helix formation and stabilization, helix nucleation and folding, helix-coil transition, and propensity of different amino acids to adopt helical motifs.31–43 In this context, several systematic investigations have been carried out to evaluate the usefulness and predictive power of different force field parameters.44–46 It is observed from different studies that there are still unanswered questions on the helix forming tendency of various amino acids and its positional dependence in a selected amino acid sequence as well as tolerance of helical sequence to accommodate other amino acids. In this investigation, an attempt has been made to study the helix forming tendency of valine and tolerance of helical motif to substitution of valine at different positions from N to C terminals using a molecular dynamics method. Computational Details The host and host-guest peptides were built in extended conformation with the ends blocked by acetyl (Ace) and * To whom correspondence should be addressed. Tel: +91 44 24411630. Fax: +91 44 24911589. E-mail: [email protected].

SCHEME 1: Schematic Representation of Various Model Peptides

N-methyl (Nme) groups as described in Scheme 1. The molecular dynamics simulation on these peptides was carried out in vacuum using the ff99SB force field employing the Amber 9 suite of programs.47 Side chains were minimized for 1000 cycles in the steepest descent algorithm followed by 10 000 cycles in the conjugate gradient method to reduce the initial strain in the model peptides. Finally the entire model system was minimized without any constraints for 10 000 cycles using the conjugate gradient method. Molecular dynamics simulations were performed in NVT (number of particles, volume, and temperature are kept constant) ensemble (canonical ensemble) at 300 K. All model peptides were equilibrated for 25 ps. The fluctuations in the potential and total energies confirmed the appropriate equilibration of the peptides. After equilibration, 20 ns molecular dynamics simulations were carried out on these model peptides. Bonds involving hydrogen atoms were constrained with the SHAKE algorithm using a geometrical tolerance of 0.000001 Å. A

10.1021/jp7119813 CCC: $40.75  2008 American Chemical Society Published on Web 07/03/2008

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Figure 1. Average potential energy of various model host-guest peptides.

TABLE 1: Average Radius of Gyration and End-to-End Distance for the Model Host and Host-Guest Peptides system

end to end distance [Å] average

radius of gyration Rg average

PA V1 V2 V3 V4 V5 V6 V7 V8 V9 V10

16.31 (1.56) 16.10 (1.77) 16.39 (1.49) 15.84 (1.49) 15.62 (1.37) 15.51 (3.10) 16.68 (1.45) 5.22 (0.44) 17.00 (1.47) 16.50 (1.58) 16.77 (1.50)

5.50 (0.49) 5.75 (0.18) 5.50 (0.47) 5.36 (0.48) 5.26 (0.41) 5.59 (0.51) 5.65 (0.48) 4.51 (0.110 5.84 (0.46) 5.64 (0.48) 5.62 (0.48)

cut off distance of 12 Å was used for nonbonded interactions. The nonbonded list was updated for every 20 steps. Simulations were performed with a time step of 2 fs, with exchange attempts occuring every 1 ps. Simulations were carried out in weak temperature coupling algorithm with a time constant of 0.1 ps. All the analyses were carried out using Ptraj package. Results and Discussion The guest residue, valine, is substituted in host peptide starting from N terminal to C terminal. The host-guest peptide substituted with valine in the nth position is denoted as Vn. The models of host-guest peptides are schematically represented in Scheme 1. Average potential energy obtained from 20 ns molecular dynamics simulation for all the host-guest peptides are presented in Figure 1. Average potential energy is minimum for V3 and V4 peptides. This indicates that substitution of valine in the third (V3) and fourth (V4) positions from the N-terminal of the poly-alanine is the energetically favorable. On the other hand substitution of valine in third (V8) and fourth (V7) positions from the C-terminal of the poly-alanine is energetically unfavorable. These results clearly demonstrate the positional preference of valine in helical structure. With a view to gain insight into the role of valine in the folding of host and host-guest peptides, the radius of gyration and end-to-end distance have been computed from the molecular dynamics trajectory. The calculated values are plotted in the Supporting Information, Figures 1 and 2. The average end-toend distance and radius of gyration of the peptides are presented Table 1. The radius of gyration and end-to-end distances are interrelated parameters and directly provide information about folding of peptides. As the peptides fold, the end-to-end distance decreases and, hence, the compactness of the peptide increases.

These variations can be clearly seen from the plots in the Supporting Information, Figures 1 and 2, and the results presented in Table 1. From the periodic fluctuations in the radius of gyration and end-to-end distance, it is possible to observe that both R and 310 helical conformations coexist in these model peptides due to the dynamical nature of hydrogen bonding interaction between i and i+4 and i and i+3 which is in accordance with the previous molecular dynamics and experimental studies on poly alanine peptides.38 In order to understand the effect of solvent on the coexistence of R helix and 310 helix, MD simulations were carried out using generalized Born model.48 The results obtained from the implicit solvent and gas phase simulations were compared and presented in the Figure 2 and in the Supporting Information, Table 1. It is evident from the results that both R and 310 helices coexist in solvent medium. However, the presence of solvent environment tends to increase the rate of transition between two states. Substitution of valine in first (V1), second (V2), fourth (V4), and fifth (V5) position from the N-terminal shifts the dynamical equilibrium toward the 310 conformation whereas substitution of the same in the fifth (V6) position from the C-terminal moves the dynamical equilibrium toward the R-helical conformation. The presence of valine in the third (V3) position from the N-terminal does not have any effect. In the case of host-guest peptides with valine in first (V10) and second (V9) positions from the C-terminal induces the rate of the transition between the two conformations, whereas the presence of valine in the third (V8) position from the C-terminal decreases the rate of transition. It is observed from the radius of gyration and end-toend distance that the valine in the fourth (V7) position from the C-terminal does not favor helical conformation. V7 peptide tends to form globular conformation. The coexistence of these two conformations has also been substantiated by the analysis of hydrogen bonding interaction. Occupancy of each possible i...i + 3 and i...i + 4 hydrogen bonds are presented in Figures 3 and 4. When compared to i...i + 4 hydrogen bonds, the occupancy of i...i + 3 is maximum in agreement with the previous findings.38 The occupancy of hydrogen bonding interaction between i and i + 4 residues are considerably low for the V7 peptide when compared to all other valine containing model peptides. It can be noted that V4 has highest occupancy for i and i + 4 hydrogen bonds and hence corresponding tendency for R helical conformation. It is interesting to observe that the R-helix forming tendency of V4 is more than that of Ala10 in concurrence with the previous analysis of the helix found in the crystal structures database.49 It is evident from the analysis that in the four-length R helices and longer R-helices, the hydrophobic residues are more preferred than the other residues. Specifically in longer helices, phenylalanine, isoleucine, methionine, and valine are preferred in the fourth position from the N-terminal.49 Our molecular dynamics results clearly predict this observation of the formation of a helix in valine substituted poly-alanine peptides. The percentage of helical tendency for residues in the fifth and sixth position is zero for V7 peptide. These results clearly reinforce the fact that valine substitution can not be tolerated by the poly-alanine sequence in the forth position from the C-terminal. To gain further understanding, we use the Ramachandran plot for the alanine residues in the fifth and sixth position of V7 host-guest peptide (data not shown). The φ and ψ angles for the fifth residue fall in the not allowed region in the Ramachandran plot. Further analysis shows that for the fifth residue ω deviates drastically from the 180° as presented in the Supporting Information, Figure 3. For the sixth residue, φ

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Figure 2. End-to-end distance of the poly alanine model simulated in solvent and gas phase.

Figure 3. Occupancy of i and i + 3 hydrogen bonds for host and host-guest peptides.

backbone carbonyl atoms. The average structure of the host and host-guest peptide is shown in Figure 5. From the figures, it is found that substitution of valine in the poly alanine induces conformational changes. To confirm the molecular dynamics results, the helical content prediction server AGADIR has been used to simulate the circular dichorisum spectra for various model peptides.50–52 The CD spectra are presented in Figure 6. Substitution of valine in the fourth position from the C-terminal has the lowest helical content when compared to that of others. Figure 4. Occupancy of i and i + 4 hydrogen bonds for host and host-guest peptides.

and ψ occupy the region corresponding to that of the β-turn in the Ramachandran plot. As a consequence, the substitution of valine at the fourth position from the C-terminal does not favor formation of helix due to sterical hindrance. It is well-known that the first helical turn is not geometrically equivalent to that of other helical turns in the peptides.49 It can be seen from Figure 4 in the Supporting Information that the substitution of valine residue at the fourth (V4) position from the N-terminal is favored due to restriction of rotational freedom of side-chain of valine when compared to that of its presence in the other positions. In fact, the conformation g- (χ + 60°) is completely prohibited for valine at the central positions of helices because of steric hindrance between the side-chain and

Conclusion The following interesting observations emerge from the molecular dynamic investigation on the valine substituted poly alanine model peptides. (i) The positional preference of valine in the poly alanine based helix is evident from the, radius of gyration, end-to-end distance, and hydrogen bond dynamics. The coexistence of 310 and R helical conformations is evident from the molecular dynamics analysis. The solvent environment increases the rate of transition from one helical form to the other. (ii) The replacement of alanine by valine in the first (V1), second (V2), fourth (V4), and fifth (V5) positions from the N-terminal favors formation of 310 conformations. On the other hand, substitution of the same in the fifth (V6) position from

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Figure 5. Average structure of host and host-guest peptides.

(iv) It is found from the results that valine in the fourth (V7) position from C-terminal does not favor helical conformation. V7 peptide tends to form globular conformation. (v) These finding are in close agreement with the simulated CD spectra on the model peptides. These observations are useful to understand the design strategies for synthesis of new peptides with biological and pharmaceutical applications and the protein folding process in short helical peptides. Acknowledgment. We acknowledge DST and CSIR, Government of India, New Delhi for financial support. Supporting Information Available: Additional figures and an additional table. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes

Figure 6. Percentage of helicity predicted for host and host-guest peptides using AGADIR server (A) for various temperatures and (B) for various ionic strengths.

the C-terminal leads to stabilization of the R-helical conformation. The presence of valine in the third (V3) position does not have any effect on the chosen model peptide. (iii) The rate of transition between the 310 and R helical conformation is enhanced by the substitution of valine in first (V10) and second (V9) positions from the C-terminal whereas the presence of valine in the third (V8) position from the C-terminal decreases the rate of transition.

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