Redefining the dry molten globule state of proteins.

Dynamics and function of proteins are governed by the structural and energetic properties of the different states they adopt and the barriers separating them. In earlier work, native-state triplet-triplet energy transfer (TTET) on the villin headpiece subdomain (HP35) revealed an equilibrium between a locked native state and an unlocked native state, which are structurally similar but have different dynamic properties. The locked state is restricted to low amplitude motions, whereas the unlocked state shows increased conformational flexibility and undergoes local unfolding reactions. This classified the unlocked state as a dry molten globule (DMG), which was proposed to represent an expanded native state with loosened side-chain interactions and a solvent-shielded core. To test whether the unlocked state of HP35 is actually expanded compared to the locked state, we performed high-pressure TTET measurements. Increasing pressure shifts the equilibrium from the locked toward the unlocked state, with a small negative reaction volume for unlocking (ΔV(0)=-1.6±0.5cm(3)/mol). Therefore, rather than being expanded, the unlocked state represents an alternatively packed, compact state, demonstrating that native proteins can exist in several compact folded states, an observation with implications for protein function. The transition state for unlocking/locking, in contrast, has a largely increased volume relative to the locked and unlocked state, with respective activation volumes of 7.1±0.4cm(3)/mol and 8.7±0.9cm(3)/mol, indicating an expansion of the protein during the locking/unlocking transition. The presented results demonstrate the existence of both compact, low-energy and expanded, high-energy DMGs, prompting a broader definition of this state.

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

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 pressure-unfolded proteins. Both helix folding and unfolding become slower with increasing pressure. The volume changes associated with the addition of a single helical residue to a preexisting helix were obtained by comparing the experimental results with Monte Carlo simulations based on a kinetic linear Ising model. The reaction volume for adding a single residue to a helix is small and negative (-0.23 cm(3) per mol = -0.38 Å(3) per molecule) implying that intrahelical hydrogen bonds have a smaller volume than peptide-water hydrogen bonds. In contrast, the transition state has a larger volume than either the helical or the coil state, with activation volumes of 2.2 cm(3)/mol (3.7 Å(3) per molecule) for adding and 2.4 cm(3)/mol (4.0 Å(3) 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.

Testing the diffusing boundary model for the helix-coil transition in peptides.

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 ϕf-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 (ϕf = 0) in agreement with a diffusing boundary mechanism.