): The long-term objective of this research project is to enhance our understanding of protein chemistry and biology. The proposed research is focused on ribonuclease A (RNase A), a small protein that efficiently catalyzes the cleavage of the P-0 bond of RNA. Classic work on RNase A has contributed much information to protein science. In the proposed research, this wealth of information is combined with modern ideas and methods to achieve six specific aims.
The specific aims of this research project are (1) Staudinger ligation-develop a powerful new method for creating semisynthetic proteins; (2) peptide bond isosteres-determine the contribution of a main-chain NH to catalysis; (3) nonnatural modules-determine the contribution to conformational stability of disulfide bond isosteres, a covalent mimic of a hydrogen bond, a metathesis crosslink, and (3) turn mimics; (4) hydroxyurea inhibitor-create a new type of ribonuclease inhibitor; (5) function in vivo-determine the true physiological role of pancreatic ribonuclease and the cytosolic ribonuclease inhibitor protein (which binds to pancreatic ribonuclease with high affinity) by creating and analyzing knockout mice; and (6) three-dimensional structures-determine at atomic resolution the structures of the most informative protein variants created herein.
Aims 1 - 3 use RNase A as a model system to test hypotheses of broad applicability. In particular, these aims use and extend new techniques that combine recombinant DNA technology with synthetic organic chemistry to create proteins containing nonnatural functionalities of great potential utility.
Aims 4 - 6 address the most meaningful issues on the function and structure of RNase A itself. The proposed experiments use methods and ideas from organic chemistry, biochemistry, biophysics, molecular biology, and molecular genetics to provide new insights into protein chemistry and biology, and could ultimately lead to novel proteins for biomedical analyses and therapies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Jones, Warren
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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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
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
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
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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