title: Redefining the dry molten globule state of proteins.
abstract: 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.
authors.affiliation: Munich Center for Integrated Protein Science and Chemistry Department, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Neumaier
authors.affiliation: Munich Center for Integrated Protein Science and Chemistry Department, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany. Electronic address: t.kiefhaber@tum.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kiefhaber
mesh_terms: D004735:Energy Transfer; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016900:Neurofilament Proteins / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D011312:Pressure; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D016900:Neurofilament Proteins; D010446:Peptide Fragments; D011506:Proteins; C115223:villin headpiece subdomain peptide
keywords: high-pressure experiments; protein dynamics; protein folding; triplet–triplet energy transfer; villin headpiece subdomain