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Conformational state distributions of a hinge-bending enzyme studied by single-molecule FRET

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Inter-domain motions of PGK and smFRET studies


A new view has recently emerged showing that multi-domain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Foerster resonance energy transfer (smFRET) measurements with normal mode analysis and coarse grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these new ideas. From smFRET, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. With the simulations, we characterize the inter-domain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to above milliseconds. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multi-domain proteins these motions are rather fast and presumably not rate limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates and acts as a pre-state to reach active conformations by means of Brownian motions. (Gabba et al., Biophys. J. 2014, 107, 1913-1923)


Contact: Dr. Michele Cerminara


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