Biological & Soft Matter Seminar: Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET

Abstract:

Protein allostery plays a pivotal role in the regulation of virtually all biological processes including enzymatic catalysis, signal transduction, gene regulation, and cellular motility. The mechanism of allostery involves an intricate choreography of the protein’s structure and energetics.  While we now have many static pictures of the protein structure, measurements of protein energetics and dynamics are more difficult and are key to understanding protein function.  Over the last decade we have developed novel methods for measuring protein energetics and dynamics using transition metal ion FRET (tmFRET). We use genetic code expansion to introduce tiny, nonperturbing donor fluorophores and sites for metal ion labeling that, together, give us sensitivity to biological distance scales on the order of 10-25 Å. We have applied time-resolved tmFRET to develop mechanistic insights into membrane proteins including a cyclic nucleotide-gated ion channel (SthK) and a membrane-fusing GTPase (Mfn1). We show that time-resolved tmFRET can quantify both the heterogeneity of any given conformational state and the energetics that govern the distribution of a protein among conformational states, collectively known as protein dynamics.