The overall objective of this research project is to determine the role in catalysis of the amino acid residues in ribonuclease A (RNase A), and to create mutant and hybrid RNase A's of modified substrate specificity. RNase A is a protein of 124 amino acid residues that efficiently catalyzes the hydrolysis of RNA. The enzyme has a well-defined enzymology and three- dimensional structure, and has been widely used in protein folding studies. As one of the best characterized of all enzymes, RNase A is an ideal object for studying relationships between protein structure and enzyme function. This research project enlists protein engineering to elucidate catalysis by RNase A. The cDNA for RNase A has been cloned and expressed in S. cerevisiae.
The specific aims of this research project use mutant RNase A's: (1) to delineate the ability of Lys41 to stabilize the transition states of the catalytic reaction, (2) to determine whether Asp121 of the putative catalytic triad abstracts a proton from His119, or simply serves to stabilize the tautomer of His119 needed for catalysis, (3) to achieve altered substrate specificity (using random mutagenesis followed by phenotypic selection), and (4) to create a hybrid enzyme that catalyzes the hydrolysis of RNA only at the sequence dictated by an attached DNA oligonucleotide. To interpret chemically the measurements of enzyme action, the three-dimensional structure of RNase A mutants will be determined by X-ray diffraction analysis. The proposed experiments use techniques from molecular biology, biochemistry, and biophysical chemistry to test ideas on the relationship between protein structure and enzyme function. The results of these experiments may lead to insights into this relationship, as well as to the creation of novel enzymes for medicinal analyses and therapies, and for industrial processes.
| Windsor, Ian W; Palte, Michael J; Lukesh 3rd, John C et al. (2018) Sub-picomolar Inhibition of HIV-1 Protease with a Boronic Acid. J Am Chem Soc 140:14015-14018 |
| Chyan, Wen; Kilgore, Henry R; Raines, Ronald T (2018) Cytosolic Uptake of Large Monofunctionalized Dextrans. Bioconjug Chem 29:1942-1949 |
| Chyan, Wen; Raines, Ronald T (2018) Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging. ACS Chem Biol 13:1810-1823 |
| Mix, Kalie A; Lomax, Jo E; Raines, Ronald T (2017) Cytosolic Delivery of Proteins by Bioreversible Esterification. J Am Chem Soc 139:14396-14398 |
| Smith, Thomas P; Windsor, Ian W; Forest, Katrina T et al. (2017) Stilbene Boronic Acids Form a Covalent Bond with Human Transthyretin and Inhibit Its Aggregation. J Med Chem 60:7820-7834 |
| Hoang, Trish T; Smith, Thomas P; Raines, Ronald T (2017) A Boronic Acid Conjugate of Angiogenin that Shows ROS-Responsive Neuroprotective Activity. Angew Chem Int Ed Engl 56:2619-2622 |
| Burke, Eileen G; Gold, Brian; Hoang, Trish T et al. (2017) Fine-Tuning Strain and Electronic Activation of Strain-Promoted 1,3-Dipolar Cycloadditions with Endocyclic Sulfamates in SNO-OCTs. J Am Chem Soc 139:8029-8037 |
| Chyan, Wen; Kilgore, Henry R; Gold, Brian et al. (2017) Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging. J Org Chem 82:4297-4304 |
| Newberry, Robert W; Raines, Ronald T (2016) A prevalent intraresidue hydrogen bond stabilizes proteins. Nat Chem Biol 12:1084-1088 |
| Mix, Kalie A; Aronoff, Matthew R; Raines, Ronald T (2016) Diazo Compounds: Versatile Tools for Chemical Biology. ACS Chem Biol 11:3233-3244 |
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