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Item DENSITY FUNCTIONAL CALCULATIONS OF BACKBONE 15N CHEMICAL SHIELDINGS IN PEPTIDES AND PROTEINS(2009) Cai, Ling; Fushman, David; Kosov, Daniel S; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this dissertation, we describe computational and theoretical study of backbone 15N chemical shieldings in peptides and proteins. Comprehensive density functional calculations have been performed on systems of different complexity, ranging from model dipeptides to real proteins and protein complexes. We begin with examining the effects of solvation, hydrogen bonding, backbone conformation, and the side chain identity on 15N chemical shielding in proteins by density functional calculations. N-methylacetamide (NMA) and N-formyl-alanyl-X (with X being one of the 19 naturally occurring amino acids excluding proline) were used as model systems for this purpose. The conducting polarizable continuum model was employed to include the effect of solvent in the calculations. We show that the augmentation of the polarizable continuum model with the explicit water molecules in the first solvation shell has a significant influence on isotropic 15N chemical shift but not as much on the chemical shift anisotropy. The difference in the isotropic chemical shift between the standard &beta-sheet and standard &alpha-helical conformations ranges from 0.8 ppm to 6.2 ppm depending on the residue type, with the mean of 2.7 ppm. This is in good agreement with the experimental chemical shifts averaged over a database of 36 proteins containing >6100 amino acid residues. The orientation of the 15N chemical shielding tensor as well as its anisotropy and asymmetry are also in the range of values experimentally observed for peptides and proteins. Having applied density functional calculation successfully to model peptides, we develop a computationally efficient methodology to include most of the important effects in the calculation of chemical shieldings of backbone 15N in a protein. We present the application to selected &alpha-helical and &beta-sheet residues of protein G. The role of long-range intra-protein electrostatic interactions by comparing models with different complexity in vacuum and in charge field is analyzed. We show that the dipole moment of the &alpha-helix can cause significant deshielding of 15N; therefore, it needs to be considered when calculating 15N chemical shielding. We emphasize the importance of including interactions with the side chains that are close in space when the charged form for ionizable side chains is adopted in the calculation. We also illustrate how the ionization state of these side chains can affect the chemical shielding tensor elements. For &alpha-helical residues, chemical shielding calculations using a 8-residue fragment model in vacuum and adopting the charged form of ionizable side chains yield a generally good agreement with experimental data. We also performed computational modeling of the chemical shift perturbations occurring upon protein-protein or protein-ligand binding. We show that the chemical shift perturbations in ubiquitin upon dimer formation can be explained qualitatively through computation. This dissertation hence demonstrates that quantum chemical calculations can be successfully used to obtain a fundamental understanding of the relationship between chemical shielding and the surrounding protein environment for the elusive case of 15N and therefore enhance the role of 15N chemical shift measurements in the analysis of protein structure and dynamics.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.