Single Molecule Fluorescence Investigation of Protein Folding

Title of Document: SINGLE MOLECULE FLUORESCENCE INVESTIGATION OF PROTEIN FOLDING Jianwei Liu, Ph.D., 2008 Directed By: Associate Professor, Victor Muñoz, Department of Chemistry and Biochemistry In addition to the well known two-state folding scenario, the energy landscape theory of protein folding predicts the possibility of downhill folding under native conditions. This intriguing prediction was extended by Victor Muñoz and coworkers to include global downhill folding. i.e. a barrierless free energy surface and unimodal conformational distributions at all degrees of unfolding stress. A small protein, BBL, has been shown to follow this behavior as evidenced from experiments and simulations. However, the identification of BBL as a global downhill folder has raised a significant amount of controversy with some groups claiming that it still folds in a two-state fashion. The objective of this thesis is to characterize the conformational distribution of BBL using single molecule Förster resonance energy transfer (SM-FRET) to obtain direct evidence for the downhill folding in BBL. We carried out SM-FRET measurements at 279 K to slow down the protein dynamics to 150 μs thus enabling the use of a 50 μs binning time (the short binning time being a first in SM measurements). By optimizing the microscope system setup and employing a novel Trolox-cysteamine fluorophore protection system, we obtained sufficient signal to construct reliable 50 μs SM-FRET histograms. The data show clear unimodal conformational distributions at varying denaturant concentrations thus demonstrating the downhill folding nature of BBL. Further SM-FRET measurements on a two-state folder, α-spectrin SH3 produced bimodal histograms indicating that our experimental setup works well and that the unimodal distributions of BBL are not due to instrumental errors. The comparison of ensemble FRET measurements on labeled proteins (both BBL and αspectrin SH3) with CD measurements on the corresponding unlabeled proteins shows that the fluorophores do not affect the protein stability. We also simulated the expected histograms if BBL were a two-state folder using Szabo’s photon statistics theory of SM-FRET. The two-state simulation results are inconsistent with the experimental histograms even under very conservative assumptions about BBL’s relaxation time. Therefore, all the control experiments and simulations exclude any possible artifacts, which shows our results are quite robust. Additionally, we estimated the relaxation time of BBL from the histogram width analysis to be consistent with independent kinetic measurements. SINGLE MOLECULE FLUORESCENCE INVESTIGATION OF PROTEIN FOLDING