Transition state and ground state properties of the helix–coil transition in peptides deduced from high-pressure studies

Abstract

Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the α-helix, and measure triplet-triplet energy transfer at high pressure to determine volume changes associated with the helix-coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressureunfolded proteins. Both helix folding and unfolding become slower with increasing pressure. per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute.protein stability | helix dynamics | protein dynamics U nderstanding the effect of pressure on protein stability and dynamics provides insight into fundamental principles and mechanisms of protein folding (1). For most proteins, increasing pressure shifts the folding equilibrium toward the unfolded state, which, according to Le Chatelier's principle, shows that the native state has a larger volume than the unfolded state (2-4). The origin of the volume increase upon folding has been discussed controversially for a long time. The major obstacle in the interpretation of volume changes is opposing contributions from different effects. Analysis of high-resolution X-ray structures suggested that formation of intramolecular hydrogen bonds and van der Waals interactions in the native state lead to a decrease in atomic volumes and thus to a decrease in protein volume upon folding (5). Further, water around hydrophobic groups has a larger volume than bulk water, which also leads to a decrease in volume upon burial of hydrophobic groups in the native protein (6). On the other hand, formation of ordered water structures around charged groups (7) and solvation of the peptide backbone decrease the water volume (6), which leads to a volume increase upon folding. Recent experimental results suggested that volume changes associated with transfer of groups from solvent to the protein interior upon folding are small and that the formation of void volumes in native proteins is the major origin of the volume increase upon folding (8).Volume changes associated with the formation of protein folding transition states are only poorly characterized. Highpressure stopped-flow experiments on tendamistat (9) and cold shock protein (10), as well as pressure-jump experiments on coldshock protein (10) and an ankyrin repeat domain (11), revealed that volumes of protein folding transition states are close to the volume of the native...

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

“…Typically, pressure effects are attributed primarily to the elimination of internal cavities through hydration (12,69), leading to local and eventually global unfolding (20). Based on the similar helical content of apoMb and the pressurepopulated MG state, it was suggested that pressure populates alternative packing arrangements of the helical core rather than producing local unfolding to reduce internal cavity volume (13), and this notion is consistent with recent studies showing that pressure shifts helix-coil equilibria toward the helical conformation rather than an unfolded state (70,71). The high-pressure DEER data add the amplitude of conformational rearrangements involved to this model.…”

Role of solvation in pressure-induced helix stabilization

“…Below we focus on the Zimm-Bragg model, as this has been used to characterize the experimental data. 14,15 In particular, the elongation parameter s corresponds to the equilibrium constant for adding a single helical hydrogen bond.…”

“…In future, it would be very interesting to compare simulation results with experimental data for other model peptides such as β-hairpins, should such data become available. Experimental kinetics results for the pressure-dependence of helix 15 and protein folding 34,35 kinetics are also a rich source of information for future detailed comparison with molecular simulation. …”

“…Recent experimental work on the pressure dependence of the helix-coil equilibrium, using either FTIR spectroscopy 13,14 or triplet state quenching experiments 15 has in fact found the opposite trend to that for protein folding: namely a negative reaction volume for helix formation, resulting in pressure stabilization of helices for all positive pressures. The origin of this effect is not clear, especially considering the small magnitude of the volume change (≈0.2-1.0 cm 3 mol −1 per residue).…”

Conformational Substates of Amyloidogenic hIAPP Revealed by High Pressure NMR Spectroscopy

“…Our observed differences in the combined first and second order pressure coefficients reveal different transient structural states in amino acid residues 3–20 between the two peptides hIAPP(1–37) and Aβ(1–40). It is important to point out that in contrast with proteins helices in small peptides may become more stable with increasing pressure as found in experimental studies on a 21‐amino‐acid alanine‐based model peptide by Neumaier et al . This could imply a pressure populated equilibrium population shift towards the hypothetical active form of IAPP.…”