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...

The dynamics of peptide α-helices have been studied extensively for many years, and the kinetic mechanism of the helix-coil dynamics has been discussed controversially. Recent experimental results have suggested that equilibrium helix-coil dynamics are governed by movement of the helix/coil boundary along the peptide chain, which leads to slower unfolding kinetics in the helix center compared with the helix ends and position-independent helix formation kinetics. We tested this diffusion of boundary model in helical peptides of different lengths by triplet-triplet energy transfer measurements and compared the data with simulations based on a kinetic linear Ising model. The results show that boundary diffusion in helical peptides can be described by a classical, Einstein-type, 1D diffusion process with a diffusion coefficient of 2.7·10 7 (amino acids) 2 /s or 6.1·10 −9 cm 2 /s. In helices with a length longer than about 40 aa, helix unfolding by coil nucleation in a helical region occurs frequently in addition to boundary diffusion. Boundary diffusion is slowed down by helix-stabilizing capping motifs at the helix ends in agreement with predictions from the kinetic linear Ising model. We further tested local and nonlocal effects of amino acid replacements on helix-coil dynamics. Single amino acid replacements locally affect folding and unfolding dynamics with a ϕ f -value of 0.35, which shows that interactions leading to different helix propensities for different amino acids are already partially present in the transition state for helix formation. Nonlocal effects of amino acid replacements only influence helix unfolding (ϕ f = 0) in agreement with a diffusing boundary mechanism.α-helix capping | ϕ-value | protein folding

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