Protein folding: mechanism and principles

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

Protein folding is perhaps the most fundamental of all protein functions. Protein folding is a difficult process and is potentially dangerous when it goes wrong. Correct folding and misfolding are key to the function of all cells and to maintain health. Yet there is still no general agreement about how this basic process works: do proteins fold through discrete well defined intermediates in discrete pathways, as in the classical view, or do they fold by way of multiple independent unrelated pathways as in the so-called new view. This work uses a powerful new approach developed in the last award to understand the protein folding problem, and is likely to provide the insightful information to define the folding process in some detail and answer key questions in the field. These studies will engage and actively train, teach, and support postdoctoral, undergraduate, and high school students in active hands-on research at the forefront of modern structural biology. Naturally occurring protein hydrogen exchange (HX) behavior is very sensitive to the biophysical properties that allow protein molecules to accomplish their myriad biological functions. It has therefore been able to contribute importantly to protein science research, especially when measured by NMR at amino acid resolution. However NMR is limited to the study of relatively small proteins in quantity at high concentrations. Recent work in the PI's laboratory has developed an advanced mass spectrometry analysis (HX MS) that can measure HX at near amino acid resolution in large and complex protein systems in previously unreachable situations using minuscule amounts of biological material. This technique will be used to study the protein folding problem, which lies at the base of many important problems in biological science. The detailed mechanism of protein folding itself is not understood, mainly because transient folding intermediates and folding pathways are beyond the reach of the usual high resolution structural methods. The newly advanced HX MS technology is able to track the kinetic folding process and describe the intermediate forms and pathways that carry an initially unfolded protein to its final native state. In the proposed work, HX MS technology will be used to ask how proteins fold, why they fold in that way, and how helper chaperone molecules function to promote folding. The how question will be addressed in studies of a biologically typical large protein, unlike almost all previous studies (both theoretical and experimental) which have been directed at small proteins that account for only a few percent of the biological proteome. The why question will be addressed by studying at high structural resolution the folding of laboratory-designed proteins that have not been shaped and selected through biological evolution. Chaperone function will be addressed by studying the detailed folding of a protein while it is contained within the active GroEL chaperonin cavity. This work may well change the current protein folding paradigm and, more broadly, will provide a powerful new tool for protein structure-function studies that have simply not been possible before. This work is being funded by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division of BIO, and co-funded by the Chemistry of Life Processes Program in the Chemistry Division of MPS.