Physics
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Item Protein folding and amyloid formation in various environments(2008-11-21) O'Brien, Edward Patrick; Thirumalai, Devarajan; Brooks, Bernard; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Understanding and predicting the effect of various environments that differ in terms of pH and the presence of cosolutes and macromolecules on protein properties is a formidable challenge. Yet this knowledge is crucial in understanding the effect of cellular environments on a protein. By combining thermodynamic theories of solution condition effects with statistical mechanics and computer simulations we develop a molecular perspective of protein folding and amyloid formation that was previously unobtainable. The resulting Molecular Transfer Model offers, in some instances, quantitatively accurate predictions of cosolute and pH effects on various protein properties. We show that protein denatured state properties can change significantly with osmolyte concentration, and that residual structure can persist at high denaturant concentrations. We study the single molecule mechanical unfolding of proteins at various pH values and varying osmolyte and denaturant concentrations. We find that the the effect of varying solution conditions on a protein under tension can be understood and qualitatively predicted based on knowledge of that protein's behavior in the absence of force. We test the accuracy of FRET inferred denatured state properties and find that currently, only qualitative estimates of denatured state properties can be obtained with these experimental methods. We also explore the factors governing helix formation in peptides confined to carbon nanotubes. We find that the interplay of the peptide's sequence and dimensions, the nanotube's diameter, hydrophobicity and chemical heterogeneity, lead to a rich diversity of behavior in helix formation. We determine the structural and thermodynamic basis for the dock-lock mechanism of peptide deposition to a mature amyloid fibril. We find multiple basins of attraction on the free energy surface associated with structural transitions of the adding monomer. The models we introduce offer a better understanding of protein folding and amyloid formation in various environments and take us closer to understanding and predicting how the complex environment of the cell can effect protein properties.Item SINGLE MOLECULE FLUORESCENCE INVESTIGATION OF PROTEIN FOLDING(2008-09-30) liu, jianwei; Munoz, Victor; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)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 α-spectrin 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.