Kinetics of Coupled Binding and Conformational Change in Proteins and RNA

Ligand binding can modulate function of proteins and nucleic acids by changing both the populations of functionally distinct conformational states and the timescales on which they interconvert. For this reason, both thermodynamic and kinetic details of coupling can be important to proper function. How tightly does ligand bind to the different conformational states? What effect does ligand binding have on the conformational equilibrium and conformational kinetics? On what timescales and in what order do binding and conformational change occur? Using a combination of stopped-flow kinetics, isothermal titration calorimetry, and x-ray crystallography, we determine the mechanisms of coupled binding and conformational change in protein (Bacillus subtilis RNase P protein) and RNA (DP17 biosensor) systems.

The results demonstrate that rigorous kinetic analysis can be used to estimate the equilibrium and rate constants for conformational changes, as well as the affinities of ligands for different conformational states. A single ligand can bind to different conformational states of the same protein or nucleic acid with affinities that differ by orders of magnitude. This binding shifts the conformational equilibrium towards the higher affinity state through a combination of increasing rate constants for the forward conformational change and decreasing rate constants for the reverse conformational change. Using a flux-based analysis of the mechanisms we show that molecular recognition is kinetically partitioned between a number of pathways that differ by the order in which binding and conformational change occur. The absolute and relative flux through these pathways varies with ligand concentration, the affinities of the ligand for the various conformational states, and the ability of ligand to accelerate the conformational change. Together, the results give insights into how biological function depends on the kinetic and thermodynamic details of coupled binding and conformational change.