In this project, the PI will study the physical foundation of protein folding kinetics: To what extent can the widely assumed transition state theory accurately describe protein folding? To achieve this goal, the proteins lambda repressor, ubiquitin, and phosphoglycerate kinase will be modified by site-directed mutagenesis to speed up their folding kinetics into the microsecond range. Preliminary work has shown that transition state theory breaks down below 2 microseconds. This lies well within the resolution capability of the temperature-jump instrumentation used in this research. As a result, protein ensembles that are normally hidden by a folding barrier can be examined in fast-folding proteins. Preliminary indications are that the dynamics is heterogeneous. This work will quantify the heterogeneity by probing the multiple timescales and the number of such ensembles that have different spectroscopic signatures. A number of specific technologies will be developed to achieve the overall goal of dissecting protein ensembles en route to the folded state: (1) Tailoring the electric field near tryptophan residues, and inserting electron- and proton-transfer quenchers by site-directed mutagenesis, will provide unprecedented control over protein spectral properties, allowing different ensembles to be probed in parallel by fast temperature jumps. (2) Further development of multi-channel infrared, absorption, and multi-channel fluorescence techniques will further dissect different ensembles by allowing large numbers of spectroscopic probes to be applied in parallel on the microsecond and faster time scales. (3) The role of water in the heterogeneous dynamics will be probed by reducing the water density, and by monitoring protein stability and folding rates at the resulting negative pressure. Water is often neglected in simple folding models, but de-solvation and "drying" of the native protein core upon folding are currently little understood, yet are processes of fundamental importance. The main goal of this research is a rigorous test of new statistical theories of folding, whose predictions differ from classical models most dramatically on fast time scales. Fundamental elements of the theory, such as stretching coefficients during barrier-free folding, glassy dynamics, and transition state prefactors will be measured directly for the first time.
The classical biochemical view of protein folding - a sequence of intermediate states separated by barriers culminating in the native state - has been challenged by an approach based on statistical mechanics. In this "New View," the protein traverses a rough multidimensional energy landscape on the way to the native state. Many protein ensembles, not necessarily lying on a one-dimensional path, can be explored during folding. This work will apply many folding probes in parallel to reveal how proteins move about on the multidimensional energy landscape. Cooperation with theorists will be a central feature of the work: both computational and analytical theories are now capable of determining the outlines of the energy surface, but most of the results have not been directly tested experimentally. The work will train graduate and undergraduates students. In addition, a program to bring biophysical principles into the rigorous physical chemistry and physics courses taken by majors will be completed. Currently, biological ideas often show up mainly in "watered-down" non-major courses, depriving physics and physical chemistry majors of this important field of application during their undergraduate training.
