The Unfolded State of the Villin Headpiece Helical Subdomain: Computational Studies of the Role of Locally Stabilized Structure

Abstract

The 36 residue villin headpiece helical subdomain (HP36) is one of the fastest cooperatively folding proteins, folding on the microsecond timescale. HP36‘s simple three helix topology, fast folding and small size have made it an attractive model system for computational and experimental studies of protein folding. Recent experimental studies have explored the denatured state of HP36 using fragment analysis coupled with relatively low-resolution spectroscopic techniques. These studies have shown that there is apparently only a small tendency to form locally stabilized secondary structure. Here, we complement the experimental studies by using replica exchange molecular dynamics with explicit solvent to investigate the structural features of these peptide models of unfolded HP36. To ensure convergence, two sets of simulations for each fragment were performed with different initial structures, and simulations were continued until these generated very similar final ensembles. These simulations reveal low populations of native-like structure and early folding events that cannot be resolved by experiment. For each fragment, calculated J-coupling constants and helical propensities are in good agreement with experimental trends. HP-1, corresponding to residues 41 to 53 and including the first α-helix, contains the highest helical population. HP-3, corresponding to residues 62 through 75 and including the third α-helix, contains a small population of helical turn residing at the N terminus while HP-2, corresponding to residues 52 through 61 and including the second α-helix, formed little to no structure in isolation. Overall, HP-1 was the only fragment to adopt a native-like conformation, but the low population suggests that formation of significant structure only occurs after formation of specific tertiary interactions.

Introduction

Structure in the unfolded state may play a significant role in the rapid folding of proteins by limiting the conformational search. Recent experimental work from the Fersht and Oas laboratories has highlighted the role of unfolded state structure in the rapid folding of helical proteins.1., 2. Other work has suggested the importance of polyproline II conformation (PPII) structure in the unfolded ensemble.3., 4., 5., 6. Unfortunately, direct experimental studies of the unfolded state are difficult because the most relevant unfolded state is that which is in equilibrium with the folded state under native conditions. The normal high cooperativity of folding together with the free energy balance of folding means that this state is only sparsely populated at equilibrium. Experimental difficulties also arise because of the short lifetime of the denatured state in refolding experiments. Consequently, indirect methods have to be employed but many approaches fail to examine the unfolded state under equilibrium conditions.7., 8., 9., 10., 11.

One indirect approach to studying the denatured state under native conditions is to analyze peptide fragments corresponding to elements of secondary structure derived from the whole protein. Peptide fragment analysis provides the local propensity for secondary structure formation and a potential glimpse at structures that may form in the early stages of folding. Such locally stabilized structure can play a role in rapid folding by limiting the early stages of the conformational search. For example, one popular model for folding, the diffusion collision model, postulates a critical role for locally stabilized microdomains. The determination of these structural details is potentially of great importance for the folding of helical proteins.2., 12., 13.

The villin headpiece helical subdomain (HP36), the C-terminal portion of the villin headpiece, is the shortest naturally occurring sequence that has been shown to fold cooperatively (Figure 1). Its rapid folding, small size and simple topology of three helices have made this domain an extremely popular system for computational and theoretical studies.12., 14., 15., 16., 17., 18., 19., 20., 21., 22. These studies have largely focused on generation of the correct native topology and have not investigated the details of the folding mechanism or the role of residual structure in the unfolded state.

Recent experimental work has explored the possibility of residual structure in the unfolded state of HP36.²³ In that work, a set of fragments corresponding to the three α-helices were studied as well as a larger fragment containing the first two helices. None of the individual peptide fragments showed significant helical content as judged by circular dichroism (CD) spectroscopy. However, two of the helices in HP36 are quite small in fragments 1 (HP-1) and 2 (HP-2) and the CD spectra of short helices are not well understood.24., 25., 26. Thus it is not clear how best to interpret CD studies of the small helices that may be formed by these fragments, particularly when NMR studies hint at some tendency to form non-random structure. The experimentally measured ¹H-alpha chemical shift deviations from random coil (approximately 0.25 ppm upfield) observed for the HP36 fragments suggest either sparsely populated helical conformations or ring current effects in HP-1 and fragment 3 (HP-3). These potential ambiguities are due to the limitations of the experimental methods.

Simulations can help overcome these limitations and allow for the observation of structure at the level of individual molecules instead of the ensemble averages typically provided by experiments. Computational studies can also provide atomic level detail concerning specific interactions that may not be readily available from experimental studies of rapidly interconverting ensembles. This enhances the understanding of mechanistic details of protein folding and structure. However, conformational sampling remains a significant obstacle in molecular dynamics (MD) simulations. Generation of precise populations at equilibrium is difficult due to the protein folding time-scale being much longer than is typically accessible to simulation. Hence, the study of partially populated states through simulation is hampered by poor convergence.

Replica exchange molecular dynamics (REMD) is an enhanced sampling technique27., 28., 29. that can help overcome the limited time-scale issues, yet it remains a challenging task to obtain converged results, particularly for large systems. Many different studies have used REMD to study folding in smaller model peptide systems,30., 31., 32., 33., 34., 35. however studies of unfolded state structure have been more limited.30., 36.

Here, we analyze the same set of short fragments of HP-36 that were studied experimentally in an attempt to clarify the extent of locally stabilized secondary structure. We conducted REMD simulations using both an implicit and explicit solvent model for each fragment. The results demonstrate that explicit solvent is the more accurate approach for studying these small peptides. We find that HP-1 possesses the most native-like structure of the three fragments, and the potential role that locally stabilized structure may play in the fast folding of HP36 is discussed.