Conformational Ensemble Study of WASp Interacting Protein Using NMR and Fluorescence

Intrinsically disordered proteins (IDPs) are polypeptides lacking a well-defined structure under biologically native conditions. As a result, they are best described as an ensemble of fast-exchanging conformations. The flexible nature of IDPs allows them to adopt distinct ensembles upon binding to different partners and to carry out multiple biological functions. One such protein is WASP-interacting protein (WIP), a multi-domain polypeptide that participates in the regulation of actin cytoskeleton dynamics. Here we focus on changes in WIP conformation and dynamics in the actin-binding N-terminal region of WIP (residues 2-65), using a double-mutant actin (actin-AP) to avoid unwanted polymerization. Upon sub-stoichiometric titration of actin into the WIP(2-65) sample, the HSQC intensity profile shows the largest line broadening for residues 33-45, coinciding with the actin-binding motif-WH2. Measurement of 15N rotating frame relaxation rates for WIP(2-65) in the presence of actin showed a significant increase in relaxation attributable to an exchange contribution between free and bound conformations, specifically for residues 29-44, reflecting their presence at the binding interface. These results reflect an actin-mediated change in the conformational ensemble of WIP(2-65), and validate the affinity of double-mutant actin to WIP. Based on these results we are justified in our intentions to continue studying the conformational ensemble of bound WIP2-65 and binding-folding trajectory by uncovering the binding-induced effect of actin-AP using PRE and fluorescence methods.