Proteins can now be constructed with any desired amino acid sequence. The potential applications of this technology in health and other areas are almost unlimited. Consequently, 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. To this end, we plan to study the effect of single changes in the amino acid sequence on the conformations of the folded and unfolded states, in the conformational stability, and on the thermodynamics of folding of ribonuclease T1 (RNase T1). Our primary goal is to gain a better understanding of the folded and unfolded conformations and of the forces which contribute to the conformational stability of proteins. RNase T1 is an excellent model for protein folding studies. It is the smallest enzyme known with just 104 residues, and folds to a compact globular conformation in which the hydrophobic core is sandwiched between a 4.5 turn alpha-helix and a 4-strand anti- parallel Beta-sheet. Folding can be studied with the two disulfide bonds intact or broken, and the unfolded molecule can be studied in water at 25 degrees C both disulfide bonds broken. Site-directed mutagenesis will be used to prepare mutants designed to give insight into the contribution of hydrogen bonding, and hydrophobic and electrostatic interactions to the conformational stability of RNase T1. The conformational stability of these mutant will be measured using urea and thermal unfolding experiments. The thermodynamics of folding will be studied using a differential scanning microcalori-meter. For the most interesting mutants, the three-dimensional structure of the folded protein will be determined using x-ray crystallography (In collaboration with Drs. Wolfram Saenger and Udo Heinemann), and the structure of the unfolded protein will be studied using a variety of physical techniques. The unfolded conformations of wild type RNase T1 will be studied in detail because we have evidence that the protein retains some structure after unfolding in urea. These unfolded states will be compared with the thermally unfolded states and the unfolded states that exist under physiological conditions. The influence of the disulfide bonds on the unfolded conformations will also be studied.
| 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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