Browsing by Subject "protein stability"
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Item Experimental and Computational Studies on Protein Folding, Misfolding and Stability(2010-07-14) Wei, YunProteins need fold to perform their biological function. Thus, understanding how proteins fold could be the key to understanding life. In the first study, the stability and structure of several !-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G (PGB1) were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the !-turn conformation. Our results indicate that the structure and stability of this !?hairpin peptide can be modulated in numerous ways and thus contributes towards a more complete understanding of this important model !-hairpin as well as to the folding and stability of larger peptides and proteins. The second study revealed that PGB1 and its variants can form amyloid fibrils in vitro under certain conditions and these fibrils resemble those from other proteins that have been implicated in diseases. To gain a further understanding of molecular mechanism of PGB1 amyloid formation, we designed a set of variants with mutations that change the local secondary structure propensity in PGB1, but have similar global conformational stability. The kinetics of amyloid formation of all these variants have been studied and compared. Our results show that different locations of even a single mutation can have a dramatic effect on PGB1 amyloid formation, which is in sharp contrast with a previous report. Our results also suggest that the "-helix in PGB1 plays an important role in the amyloid formation process of PGB1. In the final study, we investigate the forces that contribute to protein stability in a very general manner. Based on what we have learned about the major forces that contribute to the stability of globular proteins, protein stability should increase as the size of the protein increases. This is not observed: the conformational stability of globular proteins is independent of protein size. In an effort to understand why large proteins are not more stable than small proteins, twenty single-domain globular proteins ranging in size from 35 to 470 residues have been analyzed. Our study shows that nature buries more charged groups and more non-hydrogen-bonded polar groups to destabilize large proteins.Item Rational design of organophosphorus hydrolase for the degradation and detection of neurotoxic pesticides and chemical warfare agents(Texas A&M University, 2007-09-17) Reeves, Tony ElvernIt is critical to consider the balance between the catalytic capabilities of an enzyme and the inherent structural stability of the protein when developing enzymes for specific applications. Rational site directed mutagenesis has been used to explore the role of residues 254 and 257 in the global stability and catalytic specificities of organophosphorus hydrolase (OPH, EC 3.1.8.1). Substitution of residues H254 and H257, which are located near the active site, had a marked effect on both the global stability and substrate specificity of the enzyme. For example, the for the double mutation CoTG????2+ H254R H257L (RL) enzyme variant was 19.6 kcal/mol, 5.7 kcal/mol less than that of the wild type enzyme. At the same time, the altered enzyme was catalytically more effective against VX and VR (Russian VX), as compared to the wild type enzyme. Limited proteolysis verified the importance of residues 254 and 257 for functional stability, evidenced by enhanced resistance to irreversible unfolding associated with thermal denaturation. It has been possible to construct third generation OPH variants, which are more stable than the wild type enzyme, with a 10 ????C increase in the apparent melting temperature (TM app), yet retained desirable catalytic properties. It appeared that aromatic stacking and cation-???? interactions involving near active site residues not only affected activity but significantly contributed to the chemical and thermal stability of OPH. Rational design was used to develop an enzyme with an optimized orientation on a catalytically active biosensor surface. In these studies, lysine side chains located on the surface of OPH were used to create attachment sites to a surface plasmon resonance sensor resulting in an ensemble of enzyme orientations. Some of these orientations could be functionally restrictive if the active site is oriented toward the sensor surface. Substitution of a lysine near the active site resulted in 20% more activity with 53% less enzyme immobilized, thus increasing the specific activity of the decorated surface 2.5 fold.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.Item The folding kinetics of ribonuclease Sa and a charge-reversal variant(Texas A&M University, 2005-02-17) Trefethen, Jared M.The primary objective was to study the kinetics of folding of RNase Sa. Wild-type RNase Sa does not contain tryptophan. A tryptophan was substituted at residue 81 (WT*) to allow fluorescence spectroscopy to be used to monitor folding. This tryptophan mutation did not change the stability. An analysis of the folding kinetics of RNase Sa showed two folding phases, indicating the presence of an intermediate and consistent with the following mechanism: D ? I ? N. Both refolding limbs of the chevron plot (abcissa = final conc. of denaturant and ordinate = kinetic rate) had non-zero slopes suggesting that proline isomerization was not rate-limiting. The conformational stability of a charge-reversed variant, WT*(D17R), of a surface exposed residue on RNase Sa has been studied by equilibrium techniques. This mutant with a single amino acid charge reversal of a surface exposed residue resulted in decreased stability. Calculations using Coulomb?s Law suggested that favorable electrostatic interactions in the denatured state were the cause for the decreased stability for the charge-reversed variant. Folding and unfolding kinetic studies were designed and conducted to study the charge-reversal effect. Unfolding kinetics showed a 10-fold increase in the unfolding rate constant for WT*(D17R) over WT* and no difference in the rate of refolding. Kinetics experiments were also conducted at pH 3 where protonation of Asp17 (charge reversal site) would be expected to negate the observed kinetic effect. At pH 3 the kinetics of unfolding of WT* RNase Sa and the WT*(D17R) mutant were more similar. These kinetic results indicate that a single-site charge reversal lowered the free energy of the denatured state as suspected. Additionally, the results showed that the transition state was stabilized as well. These results show that a specific Coulombic interaction lowered the free energy in the denatured and transition state of the charge-reversal mutant, more than in WT*. To our knowledge, this is the first demonstration that a favorable electrostatic interaction in the denatured state ensemble has been shown to influence the unfolding kinetics of a protein.