Physical chemistry of biological macromolecules: kinetics, thermodynamics, spectroscopy and structure.
Rapid unfolding and folding (RUF) domains
Protein folding is commonly assumed to be a once-in-a-lifetime process that happens after protein synthesis (with or without the assistance of chaperones) resulting in a folded protein that persists in this form until it is ultimately degraded by various cellular machinery. The last two decades of protein folding kinetic studies have established that this assumption is wrong for many protein domains that spontaneously unfold then rapidly refold millions of times during their cellular lifetimes. This rapid unfolding/folding turnover is likely an evolved trait encoded in the amino acid sequence of these domains, which we refer to as RUF domains. The biological functions of this previously unrecognized trait are currently completely undefined. Our work is aimed at understanding the sequence and structural determinants of the RUF property and its role in protein function. We are using the five-domain antibody-binding region of Staphylococcal protein A (SpA-N) as a model RUF domain protein to study all three of these areas. We are developing methods to determine the structure of SpA-N, which our dynamic NMR studies indicate consists of five stable RUF domains connected by highly flexible linkers. We have shown that the domains fold and unfold independently and have a steeply increasing gradient of stability from the N- to C-terminus. We are also measuring the secretion efficiency of SpA-N in S. aureus and correlating this with the stability and kinetics of folding. We have also developed a proteomic screen for identifying other RUF domains in the yeast proteome. Coupled binding and conformational change
There is much interest in the current literature in the mechanism of coupled binding and conformational change. Unfortunately, many of the published studies are based on faulty consideration of the basic kinetics of the elementary steps. We are using the protein subunit of the ribozyme RNase P as a system in which to develop kinetic methods and the associated models to properly describe the mechanism. These studies include stopped–flow and equilibrium fluorescence; isothermal titration calorimetry; NMR; small angle X-ray scattering and X-ray crystallography. We are also developing, in collaboration with Prof. Scott Schmidler, statistical methods for fitting our data to the complex model that describes the coupled folding and ligand binding of RNase P protein. We plan to apply these methods to study the assembly of the holoenzyme, which represents a model ribonucleoprotein complex. Conformational characterization of unfolded proteins
A structural description of unfolded proteins remains the most important unsolved theoretical and experimental challenge of the protein-folding field. This is an important problem because the unfolded state is a significantly populated form of many proteins and in some cases is the precursor of pathogenic misfolded proteins. However, the number of different structures represented in the unfolded state ensemble is truly astronomical, which makes computational enumeration or experimental characterization extremely challenging. We are developing a non-atomistic theoretical model based on helix-coil and polymer physics theories to describe the unfolded ensemble of small proteins and the experimental methods to provide distributional constraints for the model.