Protein Biophysics in Cells

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

The accumulated knowledge about protein chemistry has largely been collected in vitro in dilute solution. The next frontier involves moving beyond the test tube and into cells. The PI is performing truly in vivo studies in living bacteria by using NMR spectroscopy. In-cell NMR studies in the next three years of this project will involve three protein classes: globular proteins, partially folded proteins, and intrinsically disordered proteins. Globular proteins will be represented by chymotrypsin inhibitor 2. Partially folded proteins will be represented by apocytochrome b5. Intrinsically disordered proteins will be represented by alpha-synuclein. Preliminary data show that all these systems are amenable to in-cell NMR, including studies of amide 1H exchange and spin-relaxation. The main hypothesis is that the environment inside E. coli affects the diffusion, stability, and internal dynamics of proteins. There are three objectives. (1) Determine the effective viscosity inside cells and the size of proteins inside cells by using pulsed field gradient diffusion experiments. (2) Determine protein stability in cells by using proton/deuterium exchange experiments. (3) Determine the internal dynamics of proteins in cells by monitoring spin relaxation. Broader Impacts: This project integrates research and education by advancing discovery and understanding while promoting teaching, training, and learning. Both graduate students and undergraduates will receive research training and mentoring in this project.