Browsing by Subject "Protein Conformation"
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Item Enzymatic Digestion in Aqueous-Organic Solvents: A Mass Spectrometry-Based Approach in Monitoring Protein Conformation Changes(2013-05-08) Tuvilla, Mavreen RoseThe three dimensional structure of a protein is important for its function. When misfolded, a protein may be rendered inactive or adapt a conformation that could be toxic. Studying protein folding requires an understanding of protein conformation. Traditionally, protein conformation has been studied using x-ray crystallography and nuclear magnetic resonance (NMR). X-ray crystallography is limited in the analysis of crystallized proteins and is computationally intensive. NMR deals with proteins in solution but reports only an average of conformation and the technique severely suffers from spectral overlapping due to the thousands of resonances of the protein. More recently, mass spectrometry has been employed not only to elucidate primary structures but also gather information on the three-dimensional conformation of proteins. In this study, a mass spectrometric-based approach is used to study the changes in conformation of cytochrome c and the green fluorescent protein when subjected to aqueous-organic solvent systems. The technique involved trypsin digestion and generation of peptide mass maps. For cytochrome c, the experiments were done with ethanol, methanol and acetonitrile to gain insights on naturation and denaturation. An apparent solvent effect to the rate of digestion and propensity for missed cleavages attributed to weakening of hydrophobic interactions and strengthening of intramolecular hydrogen bonding was observed. For the green fluorescent protein, sulfolane, a known supercharging agent, was used to gain insights on the effect of supercharging to protein conformation. Addition of 2.0% sulfolane shifted the charge state envelope of the protein towards lower m/z while adding lower amounts of sulfolane enhanced lower charge states while broadening the charge state envelope. The time course study showed different patterns of digestion dependent on solvent conditions implying changes in conformation. Furthermore, absorbance and fluorescence measurements suggested that addition of sulfolane protects the fluorophore from quenching. The activity of trypsin is not affected by addition of low amounts of sulfolane.Item Optimization and Analysis of Weighted-Window Predictors of Structural Disorder in Proteins(2007-5-22) Holladay, Nathan Brent; Otwinowski, ZbyszekX-ray crystallographic protein structures often contain disordered regions that are observed as missing electron density. We have developed single sequence and profile-based weighted-window predictors of structural disorder in proteins, as well as a simple method for addressing disorder-prone chain termini in disorder prediction. Optimizing the parameters for these relatively simple predictors with crystallographic data using a simulated annealing type algorithm, we achieve performance similar to that of DISOPRED2. Optimized parameters from these disorder predictors provide information relating to physical processes underlying crystallographic disorder. Optimized score adjustment values suggest a simple, monotonic relationship between disorder and residue distance from termini that is nearly the same for amino- and carboxy-terminal positions. Residue disorder parameters are strongly associated with scales from certain experimental model systems that primarily reflect hydrophobic interactions. Our data do not suggest a strong association between crystallographic disorder and secondary structure beyond that explained by hydrophobicity. Our results lend support to the idea that while hydrophobic side chain interactions are primarily involved in determining stability of the folded conformation, hydrogen bonding and similar polar interactions are primarily involved in conformational and interaction specificity.