Browsing by Subject "circular dichroism"
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Item Probing the denatured state ensemble with fluorescence(Texas A&M University, 2004-09-30) Alston, Roy WillisTo understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, ?max for the proteins and model compounds differed very little, 349.3 ? 1.2 nm. However, significant differences were observed in the fluorescence intensity at ?max (IF), suggesting that IF is more sensitive to the immediate environment than ?max. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.Item Secondary Structural and Functional Studies of Rotavirus NSP4 and Caveolin-1 Peptide-Peptide Interactions(2011-02-22) Schroeder, Megan ElizabethThe rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function.Item The Effects of Buried Ionizable Amino Acids on the Stability of Ribonuclease Sa(2014-11-07) Everett, Anthany LaurenceThe aim of this study was to investigate the stability contribution of buried ionizable amino acids in proteins. To study the stability contribution of a naturally occurring buried aspartic acid, two stabilized forms of RNase Sa designated 7S and 8S were used. In 7S, aspartic acid 79 has an elevated pK of 7.4 due to its location in the hydrophobic protein core. The stability contribution of this buried anion was calculated by comparing the ?(?G) of 7S at pH 6, 7, and 9 with that of 8S. The stability contribution of ionized Asp79 in RNase Sa 7S is estimated to be -1.8 kcal/mol. To investigate the stability contribution of non-native buried ionizable groups, we introduce aspartic acid, lysine, and alanine residues at positions 70, 71, and 92 in 7S and 8S, and measure the change in stability, ?(?G). Positions 70 and 92 are in close proximity to Asp 79, whereas position 71 is further away and partially shielded by a ?-sheet. All mutants were less stable than the parental protein, and the magnitude of the stability change is dependent on the specific location in the protein. Since structural changes can account for differences in the environment of buried charges, it is important to determine whether buried charge mutations alter the structure of our mutant proteins. To date, the structures of the 7S I71A and 8S I71D variants have been resolved by X-ray crystallography. Using software to align crystal structures based on geometries of the residue side-chains, we find that 7S I71A and 8S I71D are comparable in structure to both RNase Sa WT and to each other. Crystal structure analysis indicates that the ionizable groups of the mutant residues are isolated from aqueous solvent. The differences in stabilities of variants were measured in 7S and 8S over a pH range to determine pK values of the mutant ionizable residues. In instances where the pK of buried ionizable mutant side chains are shifted, there is an apparent positive correlation between the magnitude of the pK shift and the magnitude of the change in stability. Thus, the buried ionizable mutants that are the least likely to be charged at physiological pH were observed to have the largest stability contribution. The calculated pK values were then used to assign charge values to the ionizable groups. Once charge values were assigned, the stability contribution of electrostatic interactions was calculated using Coulomb?s law. We calculated the difference in stabilities due to electrostatic effects in the presence or absence of Asp79 in 7S and 8S, respectively. Coulombic interactions were estimated in a range between -0.9 ? 1.8 kcal/mol. Lastly, we investigate the localized effect of buried ionizable mutants on the dielectric constant. We find that introducing buried Asp mutants in 7S increases the dielectric constant, whereas making buried Lys mutations decreases the dielectric constant at each location.