Stopped-flow-time-resolved SAXS for studies of ligand-driven protein dimerization.

Protein function is highly dependent on conformational changes and association or dissociation into numerous oligomeric states. Stopped-flow approaches are suitable for probing transient kinetics in proteins, and combining this approach with small-angle X-ray scattering offers an excellent probe into the structural kinetics of protein function. In this chapter we describe in detail the methodological aspects of our recent investigation of ATP-driven dimerization of nucleotide-binding domains from the bacterial transporter MsbA using stopped-flow small-angle X-ray scattering experiments. Despite extensive studies into the structure and function of MsbA, the structural-temporal insights into the conformational rearrangements and transient intermediates along the pathway in this transporter are missing. In our stopped-flow experiments we observe the rapid formation of a transient protein dimer and subsequent dimer decay over hundreds of seconds. Thus, this approach can be used to detect kinetic parameters associated with conformational changes over a wide range of time-scales for soluble and membrane proteins.