QB3 Postdoc Seminar
Seminar | March 2 | 4:30-5:30 p.m. | 177 Stanley Hall
Speaker: Shion An Lim (Susan Marqusee lab)
Investigating the evolution of protein biophysical properties by ancestral sequence reconstruction
How do properties of proteins evolve over time? The biophysical properties of proteins affect a protein's biological function and fitness and are encoded by their amino acid sequence. Therefore, we can imagine that these properties will change over time in response to evolutionary pressures and selection. Actually studying this process is difficult, especially in understanding the historical context that led to the diversity of proteins that we exist today. To do this, we used Ancestral Sequence Reconstruction (ASR) on the biophysically well-characterized ribonuclease H (RNase H) family to infer the evolutionary history between two homologs with different stabilities. By characterizing the ancestral proteins spanning the lineages of these two homologs, we gained insight into how properties such as stability, folding kinetics, and conformations change or are maintained over evolutionary time. Additionally, we used pulsed-labeling HX-MS to obtain a stepwise folding trajectory at near-site resolution of the RNase H folding pathway across evolutionary history, which yielded mechanistic insights into how the amino acid sequence dictates the energy landscape of proteins.
Speaker: Jean Chung (Jay Groves lab)
Switch-like activation of Brutons tyrosine kinaseby membrane-mediated dimerization
The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that protein-ligand chemical affinities alone generally result in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Brutons tyrosine kinase (Btk) Using fluorescence correlation spectroscopy (FCS) and membrane-binding kinetic measurements, we identify a self-contained phosphatidylinositol (3,4,5)-trisphosphate (PIP3) sensing mechanism that achieves switch-like sensitivity to PIP3 levels, surpassing the intrinsic affinity discrimination of PIP3:PH binding. This mechanism employs multiple PIP3 binding as well as dimerization of Btk on the membrane surface. Mutational studies in live cells confirm that this mechanism is critical for activation of Btk in vivo. These results demonstrate how a single protein module can institute a minimalist coincidence detection mechanism to achieve high-precision discrimination of ligand concentration.