Linking Enzyme Conformational Dynamics To Catalytic Function With Single-molecule FRET

Abstract

of the activation energy barrier, and Dx U, the distance to the transition state. For all protecting osmolytes we measure DG U increases, demonstrating that the I27 protein is stabilized. More striking is the measurement of Dx U . Unfolding the I27 protein in water gives a Dx U ¼ 2.5 Å , a distance similar to the size of a water molecule. Water molecules have been identified as integral components of the unfolding transition state of the I27 protein, forming a solvent bridge between two b-strands. By varying osmolyte molecule size we rigorous test this solvent bridging hypothesis. We find that Dx U correlates with osmolyte size for molecules ranging in size from 2.5 Å to 5.6 Å . However, for larger molecules (> 5.6 Å ) Dx U remains unchanged relative to the value measured in water, suggesting these osmolytes do not participate in solvent bridging in the transition state. These studies uniquely probe the length scales over which solvent molecules can modify the molecular architecture of the unfolding transition of a protein, an area which remains beyond the reach of other experimental techniques.