Proteins can not be constructed with any amino acid sequence. The potential applications of this technology in health and other areas are almost unlimited. Thus, it is essential that we learn to predict how changes in the amino acid sequence will affect the function, folding, and stability of a protein. The primary goal of our research is to gain a better understanding of the forces that contribute to protein stability. To this end, we plan to continue our studies of the effect of single changes in the amino acid sequence on the conformations of the folded and unfolded states, and on the thermodynamics of folding of three related proteins: ribonuclease Sa, T1, and Ba (RNases Sa, T1 and Ba). (RNase Ba is also known as barnase.) They contain 96, 104 & 110 residues; 1, 2 & 0 disulfide bonds; and have similar three-dimensional structures. Mutants of RNases T1 & Ba will be studied to gain further insights into the contribution of hydrogen bonding and polar group burial to the conformational stability. The folding of RNase Sa will be investigated in detail, and mutants will be prepared that correspond to those already characterized in RNases T1 & Ba. We hope to gain a better understanding of the effect of local environment, i.e., the context, on the contribution of a given interaction to the stability. High resolution structures of the mutant proteins are essential for interpreting these results, and will be determined by x-ray crystallography. NMR, size-exclusion chromatography, and solvent perturbation difference spectroscopy will be used to gain a better understanding of the unfolded states of these proteins. The interactions in 8 M urea and 6 M GdnHC1 that cause RNase T1 to unfold less completely than RNase Ba will be identified, and investigated further in the unfolded states that exist under physiological conditions. Finally, a Ser 35-- Met mutants of RNase T1 has been prepared to allow cleavage of the molecule into two fragments: 1-35 containing the a-helix, and 36-104 containing most of the B-sheet. These fragments will be characterized in isolation, when combined under reducing conditions so that disulfide bonds will not form, and when the disulfide bonds are intact.
| Nick Pace, C; Scholtz, J Martin; Grimsley, Gerald R (2014) Forces stabilizing proteins. FEBS Lett 588:2177-84 |
| Pace, C Nick; Fu, Hailong; Lee Fryar, Katrina et al. (2014) Contribution of hydrogen bonds to protein stability. Protein Sci 23:652-61 |
| Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee et al. (2011) Contribution of hydrophobic interactions to protein stability. J Mol Biol 408:514-28 |
| Fu, Hailong; Grimsley, Gerald; Scholtz, J Martin et al. (2010) Increasing protein stability: importance of DeltaC(p) and the denatured state. Protein Sci 19:1044-52 |
| Nick Pace, C; Huyghues-Despointes, Beatrice M P; Fu, Hailong et al. (2010) Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins. Protein Sci 19:929-43 |
| Grimsley, Gerald R; Scholtz, J Martin; Pace, C Nick (2009) A summary of the measured pK values of the ionizable groups in folded proteins. Protein Sci 18:247-51 |
| Scholtz, J Martin; Grimsley, Gerald R; Pace, C Nick (2009) Solvent denaturation of proteins and interpretations of the m value. Methods Enzymol 466:549-65 |
| Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas et al. (2009) Increasing protein stability by improving beta-turns. Proteins 77:491-8 |
| Alston, Roy W; Lasagna, Mauricio; Grimsley, Gerald R et al. (2008) Peptide sequence and conformation strongly influence tryptophan fluorescence. Biophys J 94:2280-7 |
| Trevino, Saul R; Scholtz, J Martin; Pace, C Nick (2008) Measuring and increasing protein solubility. J Pharm Sci 97:4155-66 |
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