Stopped-Flow Kinetic Techniques for Studying Binding Reactions of Intrinsically Disordered Proteins

Intrinsically disordered proteins are abundant in signaling processes such as transcription. Suitable binding and unbinding rates of proteins with their partners are critical for allowing them to perform their biological roles. Understanding how these are achieved, and indeed designing strategies for intervening or modulating related biological processes, therefore requires kinetic studies. In this chapter, we describe stopped-flow-based methods for determining association and dissociation rate constants for pairs of macromolecular binding partners. We describe how to select the simplest appropriate model to describe the interaction, and highlight cases where it is possible to distinguish between induced fit and conformational selection binding mechanisms. Finally, we go on to describe methods for examining the role of electrostatic forces in binding processes, and for describing the transition state for binding processes that have folding associated with them.