The aim of this study is to further our understanding of the forces that contribute to protein stability and to investigate how site-directed mutagenesis might be used for increasing protein stability. Eleven proteins ranging from 36 to 370 residues have been studied here. A 36-residue VHP and a 337-residue VlsE were used as model systems for studying the contribution of the hydrophobic effect on protein stability. Mutations were made in both proteins which replaced bulky hydrophobic side chains with smaller ones. All variants were less stable than their wild-type proteins. For VHP, the destabilizing effects of mutations were smaller when compared with similar mutations reported in the literature. For VlsE, a similarity was observed. This different behavior was investigated and reconciled by the difference in hydrophobicity and cavity modeling for both proteins. Therefore, the stabilizing mechanism of the hydrophobic effect appears to be similar for both proteins. Eight proteins were used as model systems for studying the effects of mutating non-proline and non-glycine residues to statistically favored proline and glycine residues in ?-turns. The results suggest that proline mutations generally increase protein stability, provided that the replaced residues are solvent exposed. The glycine mutations, however, only have a stabilizing effect when the wild-type residues have ?, ? angles in the L? region of Ramachandran plot. Nevertheless, this strategy still proves to be a simple and efficient way for increasing protein stability. Finally, using a combination of eight previously identified stabilizing mutations; we successfully designed two RNase Sa variants (7S, 8S) that have both much higher Tms and conformational stabilities than wild-type protein over the entire pH range studied. Further studies of the heat capacity change upon unfolding (?Cps) for both proteins and their variants suggest that residual structure may exist in the denatured state of the 8S variant. An analysis of stability curves for both variants suggests that they achieve their stabilization through different mechanisms, partly attributed to the different role of their denatured states. The 7S variants may have a more rigid denatured state and the 8S variant may have a compact denatured state in comparison with that of wild-type RNase Sa.

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.

No / The conformational propensities of amino acids are an amalgamation of sequence effects, environmental effects and underlying intrinsic behavior. Many have attempted to investigate neighboring residue effects to aid in our understanding of protein folding and improve structure prediction efforts, especially with respect to difficult to characterize states, such as disordered or unfolded states. Host-guest peptide series are a useful tool in examining the propensities of the amino acids free from the surrounding protein structure. Here, we compare the distributions of the backbone dihedral angles (φ/ψ) of the 20 proteogenic amino acids in two different sequence contexts using the AAXAA and GGXGG host-guest pentapeptide series. We further examine their intrinsic behaviors across three environmental contexts: water at 298 K, water at 498 K, and 8 M urea at 298 K. The GGXGG systems provide the intrinsic amino acid propensities devoid of any conformational context. The alanine residues in the AAXAA series enforce backbone chirality, thereby providing a model of the intrinsic behavior of amino acids in a protein chain. Our results show modest differences in φ/ψ distributions due to the steric constraints of the Ala side chains, the magnitudes of which are dependent on the denaturing conditions. One of the strongest factors modulating φ/ψ distributions was the protonation of titratable side chains, and the largest differences observed were in the amino acid propensities for the rarely sampled αL region. / NIH