Determining the the Effects of Disorder on Binding Affinity

Abstract

It has long been believed that proteins require a defined three-dimensional structure to perform their specific functions. However, a class of proteins called intrinsically disordered proteins has been identified that do not require a stable structure to perform their functions. These proteins play important roles in many diverse biological processes including signal transduction, transcription, and cell division. Therefore, understanding how these proteins recognize and bind to other proteins to perform their functions is an important question. FlgM is an 88-residue intrinsically disordered protein from bacteria that regulates flagella synthesis by binding the RNA transcription factor Sigma 28. When FlgM is bound to Sigma 28, it inhibits transcription of the genes encoding the late flagella proteins. The FlgM protein is an interesting IDP since FlgM genes from different bacteria exhibit different degree of disorder region. Specifically, our lab has shown that the FlgM gene from A. aeolicus is significantly more ordered than the S. typhimurium FlgM. It is predicted that the more ordered the protein, the higher the affinity of the FlgM for Sigma 28. We are using a combination of Isothermal Titration Calorimetry (ITC) and fluorescence to determine the equilibrium binding constant and the binding kinetics for FlgM binding to Sigma 28 using proteins from a series of different bacteria, including A. aeolicus, S. typhimurium, E. coli, P. aeruginosa, and B. subtilis.