Source: UNIV OF WISCONSIN submitted to
CHEMICAL BIOLOGY OF PROTEINS
Sponsoring Institution
State Agricultural Experiment Station
Project Status
TERMINATED
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0176095
Grant No.
(N/A)
Project No.
WIS04075
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 1997
Project End Date
Sep 30, 2011
Grant Year
(N/A)
Project Director
Raines, R. T.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
BIOCHEMISTRY
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30570101000100%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
The overall goal of the proposed research is to reveal how ribonucleases promotetumor cell death.
Project Methods
The approach is to use ribonuclease A, the best characterized ribonuclease, as atemplate for the construction of hybrid proteins. Because RNase A lacks cytotoxic activity, any such activity in a hybrid protein would reveal a biochemical property necessary to cytotoxicity.

Progress 07/01/97 to 09/30/11

Outputs
OUTPUTS: The amino-acid sequence of a protein encodes its three-dimensional structure, and this structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, the Raines group is illuminating in atomic detail both the chemical basis and the biological purpose for protein structure and protein function. The efforts of my research group are leading to insights into the relationship between amino-acid sequence and protein function (or dysfunction), as well as to the creation of novel molecules with desirable properties. PARTICIPANTS: Ronald T. Raines; Sean Johnston, John Lukesh, Christine Bradford, Chelcie Eller, Joelle Lomax, Ben Caes, Mike Palte, Sayani Chattopadhyay, Amit Choudhary, Mike Levine, Nadia Sundlass, Greg Ellis, Lauren Carroll, Tzu-Yuan Chao, Rex Watkins, Ho-Hsuan Chou, Katrina Jensen, Langdon Martin, Nick McGrath, Ismet Tanrikulu, Greb Jakubczak TARGET AUDIENCES: Cancer Patients, Scientists, the Community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
My research group has discovered an RNA-cleaving enzyme that is in a human clinical trial as an anti-cancer agent, provided fundamental insight on the stability of collagen and other proteins, and developed processes to synthesize proteins and convert crude biomass into useful fuels and chemicals.

Publications

  • Stereoelectronic and steric effects in side chains preorganize a protein main chain. Matthew D. Shoulders, Kenneth A. Satyshur, Katrina T. Forest, and Ronald T. Raines (2010). Proceedings of the National Academy of Sciences USA 107, 559-564.
  • Fermentable sugars by chemical hydrolysis of biomass. Joseph B. Binder and Ronald T. Raines (2010). Proceedings of the National Academy of Sciences USA 107, 4516-4521.
  • n to Pi* Interaction and n)(Pi Pauli repulsion are antagonistic for protein stability. Charles E. Jakobsche, Amit Choudhary, Scott J. Miller, and Ronald T. Raines (2010). Journal of the American Chemical Society 132, 6651-6653.
  • Mechanistic insights on the conversion of sugars into 5-hydroxymethylfurfural. Joseph B. Binder, Anthony V. Cefali, Jacqueline J. Blank, and Ronald T. Raines (2010). Energy & Environmental Science 3, 765-771.
  • A stereoelectronic effect in prebiotic nucleotide synthesis. Amit Choudhary, Kimberli J. Kamer, Matthew W. Powner, John D. Sutherland, and Ronald T. Raines (2010). ACS Chemical Biology 5, 655-657.
  • n to Pi* Interactions in proteins. Gail J. Bartlett, Amit Choudhary, Ronald T. Raines, and Derek N. Woolfson (2010). Nature Chemical Biology 6, 615-620.
  • Modulation of an n to Pi* interaction with alpha-fluoro groups. Amit Choudhary, Charles G. Fry, and Ronald T. Raines (2010). ARKIVOC 251-262.
  • The aberrance of the 4S diastereomer of 4-hydroxyproline. Matthew D. Shoulders, Frank W. Kotch, Amit Choudhary, Ilia A. Guzei, and Ronald T. Raines (2010). Journal of the American Chemical Society 132, 10857-10865.
  • Antitumor activity of ribonuclease multimers created by site-specific covalent tethering. Thomas J. Rutkoski, John A. Kink, Laura E. Strong, Christine I. Schilling, and Ronald T. Raines (2010). Bioconjugate Chemistry 21, 1691-1702.
  • Synthesis of furfural from xylose and xylan. Joseph B. Binder, Jacqueline J. Blank, Anthony V. Cefali, and Ronald T. Raines (2010). ChemSusChem 3, 1268-1272.
  • Oligomers of a 5-carboxy-methanopyrrolidine Beta-amino acid. A search for order. Grant R. Krow, Nian Liu, Matthew Sender, Guoliang Lin, Ryan Centafont, Philip E. Sonnet, Charles DeBrosse, Charles W. Ross, III, Patrick J. Carroll, Matthew D. Shoulders, and Ronald T. Raines (2010). Organic Letters 12, 5438-5441.
  • Cellular uptake of ribonuclease A relies on anionic glycans. Tzu-Yuan Chao, Luke D. Lavis, and Ronald T. Raines (2010). Biochemistry 49, 10666-10673.
  • Advances in bioconjugation. Jeet Kalia and Ronald T. Raines (2010). Current Organic Chemistry 14, 138-147.
  • Prolyl 4-hydroxylase. Kelly L. Gorres and Ronald T. Raines (2010). Critical Reviews in Biochemistry and Molecular Biology 45, 106-124.
  • n to Pi* Interactions in the molecules of life. Amit Choudhary and Ronald T. Raines (2010). In Peptides 2010: Tales of Peptides. Proceedings of the 31st European Peptide Symposium (Michal Lebl, Morten Meldal, Knud J. Jensen, and Thomas Hoeg-Jensen, Eds.), pp. 2-3, Prompt Scientific Publishing, San Diego, CA.
  • Carpe diubiquitin. Langdon J. Martin and Ronald T. Raines (2010). Angewandte Chemie International Edition 49, 9042-9044.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The amino-acid sequence of a protein encodes its three-dimensional structure, and this structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, our research group is illuminating in atomic detail both the chemical basis and the biological purpose for protein structure and protein function. PARTICIPANTS: Christine Bradford, Graduate Research Assistant; Lauren Carroll, Graduate Research Assistant; Chelcie Eller, Graduate Research Assistant; Joelle Lomax, Graduate Research Assistant; Ben Caes, Graduate Research Assistant; Mike Palte, Graduate Research Assistant; Sayani Chattopadhyay, Graduate Research Assistant; Amit Choudhary, Graduate Research Assistant; Mike Levine, Graduate Research Assistant; Nadia Sundlass, Graduate Research Assistant; Greg Ellis, Graduate Research Assistant; Rex Watkins, Graduate Research Assistant; Cindy Chao, Graduate Research Assistant; Ho-Hsuan Chou, Postdoctoral Research Associate; Langdon Martin, Postdoctoral Research Associate; Eddie Myers, Postdoctoral Research Associate; Greb Jakubczak, Lab Manager TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The efforts of our research group have lead to insights into the relationship between amino-acid sequence and protein function (or dysfunction), as well as to the creation of novel proteins with desirable properties.

Publications

  • Nature of amide carbonyl - carbonyl interactions in proteins. Amit Choudhary, Deepa Gandla, Grant R. Krow, and Ronald T. Raines (2009). Journal of the American Chemical Society 131, 7244 - 7246.
  • Silencing an inhibitor unleashes a cytotoxic enzyme. Kimberly A. Dickson and Ronald T. Raines (2009). Biochemistry 48, 5051 - 5053.
  • ROMP from ROMP: A new approach to graft copolymer synthesis. Matthew J. Allen, Kittikhun Wangkanont, Ronald T. Raines, and Laura L. Kiessling (2009). Macromolecules 42, 4023 - 4027.
  • Onconase cytotoxicity relies on the distribution of its positive charge. Rebecca F. Turcotte, Luke D. Lavis, and Ronald T. Raines (2009). FEBS Journal 276, 4270 - 4281.
  • Origin of the stability conferred upon collagen by fluorination. Matthew D. Shoulders, Kimberli J. Kamer, and Ronald T. Raines (2009). Bioorganic & Medicinal Chemistry Letters 19, 3859 - 3862.
  • Fluorogenic affinity label for facile, rapid imaging of proteins in live cells. Rex T. Watkins, Luke D. Lavis, Vanessa M. Kung, Georgyi V. Los, and Ronald T. Raines (2009). Organic & Biomolecular Chemistry 7, 969 - 975.
  • Structure and function of Bacillus subtilis YphP, a prokaryotic disulfide isomerase with a CXC catalytic motif. Urszula Derewenda, Tomasz Boczek, Kelly R. Gorres, Minmin Yu, Li-wei Hung, David Cooper, Andrzej Joachimiak, Ronald T. Raines, and Zygmunt S. Derewenda (2009). Biochemistry 48, 8664 - 8671.
  • 5(6)-anti-Substituted-2-azabicyclo[2.1.1]hexanes: A nucleophilic displacement route. Grant R. Krow, Ram Edupuganti, Deepa Gandla, Charles W. Ross, III, Kevin C. Cannon, and Ronald T. Raines (2009). Journal of Organic Chemistry 74, 8232 - 8242.
  • Stringency of the 2-His - 1-Asp active-site motif in prolyl 4-hydroxylase. Kelly L. Gorres, Khian Hong Pua, and Ronald T. Raines (2009). PLoS ONE 4, e7635.
  • 1,9-Bis(2-pyridyl)-1,2,8,9-tetrathia-5-oxanonane. Jeet Kalia and Ronald T. Raines (2009). Molbank 2009, M642.
  • Collagen structure and stability. Matthew D. Shoulders and Ronald T. Raines (2009). Annual Review of Biochemistry 78, 929 - 958.
  • Protein engineering with the traceless Staudinger ligation. Annie Tam and Ronald T. Raines (2009). Methods in Enzymology 462, 25 - 44.
  • Stronger and (now) longer synthetic collagen. Ronald T. Raines (2009). In Peptides for Youth: The Proceedings of the 20th American Peptide Symposium (Susan Del Valle, Emanuel Escher, and William D. Lubell, Eds.), pp. xci - xcviii, Kluwer Academic, Norwell, MA.
  • Modulating collagen triple helix stability with 4-chloro-, 4-fluoro-, and 4-methylprolines. Matthew D. Shoulders and Ronald T. Raines (2009). In Peptides for Youth: The Proceedings of the 20th American Peptide Symposium (Susan Del Valle, Emanuel Escher, and William D. Lubell, Eds.), pp. 251 - 252, Kluwer Academic, Norwell, MA.
  • Protein hyperstability by preorganization. Matthew D. Shoulders, Kenneth A. Satyshur, Katrina T. Forest, and Ronald T. Raines (2009). Proceedings of the National Academy of Sciences, USA. In Press.
  • A donor - acceptor perspective on carbonyl - carbonyl interactions in proteins. Amit Choudhary and Ronald T. Raines (2009). In Breaking Away: The Proceedings of the 21st American Peptide Symposium (Michal Lebl, Ed.). In Press.
  • A phosphine-mediated conversion of azides to diazo compounds. Eddie L. Myers and Ronald T. Raines (2009). Angewandte Chemie International Edition 48, 2359 - 2363.
  • Coulombic effects on the traceless Staudinger ligation in water. Annie Tam and Ronald T. Raines (2009). Bioorganic & Medicinal Chemistry 17, 1055 - 1063.
  • Simple chemical transformation of lignocellulosic biomass into furanics for fuels and chemicals. Joseph B. Binder and Ronald T. Raines (2009). Journal of the American Chemical Society 131, 1979 - 1985.
  • Direct and continuous assay for prolyl 4-hydroxylase. Kelly L. Gorres and Ronald T. Raines (2009). Analytical Biochemistry 386, 181 - 185.
  • Ribonuclease inhibitor regulates neovascularization by human angiogenin. Kimberly A. Dickson, Dong-Ku Kang, Young Sam Kwon, Jae Chan Kim, Peter A. Leland, Byung-Moon Kim, Soo-Ik Chang, and Ronald T. Raines (2009). Biochemistry 48, 3804 - 3806.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The amino acid sequence of a protein encodes its three-dimensional structure, and this structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, the Raines' group is illuminating in atomic detail both the chemical basis and the biological purpose for protein structure and protein function. The efforts of the Raines' group are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel molecules with desirable properties. PARTICIPANTS: Current Research Group: Joe Binder, Ben Caes, Sayani Chattopadhyay, Cindy Chao, Amit Choudhary, Greg Ellis, Kelly Gorres, Mike Levine, Dr. Langdon Martin, Nicky McElfresh, Dr. Eddie Myers, Mike Palte, Matt Shoulders, Nadia Sundlass, Rebecca Turcotte, Rex Watkins TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our findings were described in the 25 published papers and 2 issued US patents listed below.

Publications

  • Stronger and (now) longer synthetic collagen. Ronald T. Raines (2008). In Peptides for Youth: Proceedings of the 20th American Peptide Symposium (Emanuel Escher, William D. Lubell, and Susan Del Valle, Eds.), Kluwer Academic, Norwell, MA; In Press.
  • Modulating collagen triple helix stability with 4-chloro-, 4-fluoro-, and 4-methylprolines. Matthew D. Shoulders and Ronald T. Raines (2008). In Peptides for Youth: Proceedings of the 20th American Peptide Symposium (Emanuel Escher, William D. Lubell, and Susan Del Valle, Eds.), Kluwer Academic, Norwell, MA; In Press.
  • Stronger and longer synthetic collagen. Ronald T. Raines (2008). In 2007 Fall Proceedings of the Materials Research Society: Protein and Peptide Engineering for Therapeutic and Functional Materials, Volume 1062E (Michael (Seungju) Yu, Seung-Wuk Lee, Derek Woolfson, Ichiro Yamashita, and Blake Simmons, Eds.), In Press.
  • Jeremy R. Knowles (1935 - 2008). Ronald T. Raines (2008). ACS Chemical Biology 3, 262 - 264.
  • Peptides and peptidomimetics as prototypes. Helma Wennemers and Ronald T. Raines (2008). Current Opinion in Chemical Biology. In Press.
  • Cytotoxic ribonuclease variants. Ronald T. Raines, Julie C. Mitchell, and Thomas J. Rutkoski (2008). US Patent 07416875.
  • Cell-permeable green fluorescent protein. Ronald T. Raines and Stephen M. Fuchs (2008). US Patent 07452973.
  • Structural basis for catalysis by onconase. J. Eugene Lee, Euiyoung Bae, Craig A. Bingman, George N. Phillips, Jr., and Ronald T. Raines (2008). Journal of Molecular Biology 375, 165 - 177.
  • Trimethyl lock: A stable chromogenic substrate for esterases. Michael N. Levine, Luke D. Lavis, and Ronald T. Raines (2008). Molecules 13, 204 - 211.
  • 4-Chloroprolines: Synthesis, conformational analysis, and effect on the collagen triple helix. Matthew D. Shoulders, Ilia A. Guzei, and Ronald T. Raines (2008). Biopolymers 89, 443 - 454.
  • Stabilization of the collagen triple helix by O-methylation of hydroxyproline residues. Frank W. Kotch, Ilia A. Guzei, and Ronald T. Raines (2008). Journal of the American Chemical Society 130, 2952 - 2953.
  • Electronic and steric effects on the rate of the traceless Staudinger ligation. Annie Tam, Matthew B. Soellner, and Ronald T. Raines (2008). Organic & Biomolecular Chemistry 6, 1173 - 1175.
  • Genetic selection for peptide inhibitors of angiogenin. Bryan D. Smith and Ronald T. Raines (2008). Protein Engineering, Design & Selection 21, 289 - 294.
  • Practical syntheses of 4-fluoroprolines. Mukund S. Chorghade, Debendra K. Mohapatra, Gokarneswar Sahoo, Mukund K. Gurjar, Manish V. Mandlecha, Nitin Bhoite, Santosh Moghe, and Ronald T. Raines (2008). Journal of Fluorine Chemistry 129, 781 - 784.
  • Conformational preferences of substrates for human prolyl 4-hydroxylase. Kelly L. Gorres, Ram Edupuganti, Grant R. Krow, and Ronald T. Raines (2008). Biochemistry 47, 9447 - 9455.
  • Hydrolytic stability of hydrazones and oximes. Jeet Kalia and Ronald T. Raines (2008). Angewandte Chemie International Edition 47, 7523 - 7526.
  • A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells. Melissa M. Yatzeck, Luke D. Lavis, Tzu-Yuan Chao, Sunil S. Chandran, and Ronald T. Raines (2008). Bioorganic & Medicinal Chemistry Letters 18, 5864 - 5866.
  • Self-assembled collagen-like peptide fibers as templates for metallic nanowires. Daniel Gottlieb, Stephen A. Morin, Song Jin, and Ronald T. Raines (2008). Journal of Materials Chemistry 18, 3865 - 3870.
  • Coulombic effects on the traceless Staudinger ligation in water. Annie Tam and Ronald T. Raines (2008). Bioorganic & Medicinal Chemistry. In Press.
  • Design and characterization of an HIV-specific ribonuclease zymogen. Rebecca F. Turcotte and Ronald T. Raines (2008). AIDS Research and Human Retroviruses. In Press.
  • Interaction of onconase with the human ribonuclease inhibitor protein. Rebecca F. Turcotte and Ronald T. Raines (2008). Biochemical and Biophysical Research Communications. In Press.
  • A phosphine-mediated conversion of azides to diazo-compounds. Eddie L. Myers and Ronald T. Raines (2008). Angewandte Chemie International Edition. In Press.
  • Direct and continuous assay for prolyl 4-hydroxylase. Kelly L. Gorres and Ronald T. Raines (2008). Analytical Biochemistry. In Press.
  • Ribonucleases as novel chemotherapeutics: The ranpirnase example. J. Eugene Lee and Ronald T. Raines (2008). BioDrugs 22, 53 - 58.
  • Bright ideas for chemical biology. Luke D. Lavis and Ronald T. Raines (2008). ACS Chemical Biology 3, 142 - 155.
  • Evasion of ribonuclease inhibitor as a determinant of ribonuclease cytotoxicity. Thomas J. Rutkoski and Ronald T. Raines (2008). Current Pharmaceutical Biotechnology 9, 185 - 199.
  • Olefin metathesis for chemical biology. Joseph B. Binder and Ronald T. Raines (2008). Current Opinion in Chemical Biology. In Press.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: A protein is a string of amino acids that folds into a three-dimensional structure. Proteins perform the molecular functions that are necessary for life. These functions include catalysis of biochemical reactions (by enzymes), neutralization of foreign toxins (by antibodies), and stimulation of cellular activity (by hormones). The goal of our research program is to understand how the amino acid sequence of a protein encodes its three-dimensional structure, and how this structure manifests itself in function. The 17 listed publications report on the output of our efforts. TARGET AUDIENCES: Target audience would be cancer patients.

Impacts
The 17 listed publications report on the outcome of our efforts and its impact.

Publications

  • Multilayered films fabricated from an oligoarginine-conjugated protein promote efficient surface-mediated protein transduction. Jewell, C. M.; Fuchs, S. M.; Flessner, R. M.; Raines, R. T.; Lynn, D. M. Biomacromolecules 2007, 8, 857-863.
  • Arginine grafting to endow cell-permeability. Fuchs, S. M.; Raines, R. T. ACS Chem. Biol. 2007, 2, 167-170.
  • Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein. Johnson, R. J.; McCoy, J. G.; Bingman, C. A.; Phillips, G. N. Jr., Raines, R. T. J. Mol. Biol. 2007, 368, 434-449.
  • General method for site-specific protein immobilization by Staudinger ligation. Kalia, J.; Abbott, N. L.; Raines, R. T. Bioconjug. Chem. 2007, 18, 1064-1069.
  • Genetic selection reveals the role of a buried, conserved polar residue. Johnson, R. J.; Lin, S. R.; Raines, R. T. Protein Sci. 2007, 16, 1609-1616.
  • Tuning the pKa of fluorescein to optimize binding assays. Lavis, L. D.; Rutkoski, T. J.; Raines, R. T. Anal. Chem. 2007, 79, 6775-6782.
  • Cytotoxic ribonucleases: The dichotomy of Coulombic forces. Johnson, R. J.; Chao, T.-Y.; Lavis, L. D.; Raines, R. T. Biochemistry 2007, 46, 10308-10316.
  • Using measurements of anchoring energies of liquid crystals on surfaces to quantify proteins captured by immobilized ligands. Govindaraju, T.; Bertics, P. J.; Raines, R. T.; Abbott, N. L. J. Am. Chem. Soc. 2007, 129, 11223-11231.
  • Is glycine a surrogate for a D-amino acid in the collagen triple helix? Horng, J.-C.; Kotch, F. W.; Raines, R. T. Protein Sci. 2007, 16, 208-215.
  • Salicylaldimine ruthenium alkylidene complexes: Metathesis catalysts tuned for protic solvents. Binder, J. B.; Guzei, I. A.; Raines, R. T. Adv. Synth. Catal. 2007, 349, 395-404.
  • Water-soluble phosphinothiols for traceless Staudinger ligation and integration with expressed protein ligation. Tam, A.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc. 2007, 129, 11431-11430.
  • Protein prosthesis: 1,5-Disubstituted[1,2,3]triazoles as cis-peptide bond surrogates. Tam, A.; Arnold, U.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc. 2007, 129, 12670-12671.
  • Catalysis of imido-group hydrolysis in a maleimide conjugate. Kalia, J.; Raines, R. T. Bioorg. Med. Chem. Lett. 2007, 17, 6286-6289.
  • Olefin metathesis in homogeneous aqueous media catalyzed by conventional ruthenium catalysts. Binder, J. B.; Blank, J. J.; Raines, R. T. Org. Lett. 2007, 9, 4885-4888.
  • Intraspecies regulation of ribonucleolytic activity. Johnson, R. J.; Lavis, L. D.; Raines, R. T. Biochemistry 2007, 46, 13131-13140.
  • Increasing the potency of a cytotoxin with an arginine graft. Fuchs, S. M.; Rutkoski, T. J.; Kung, V. M.; Groeschl, R. T.; Raines, R. T. Protein Eng. Des. Sel. 2007, 20, 505-509.
  • Hyperstable collagen based on 4-fluoroproline residues. Raines, R. T. In Current Fluoroorganic Chemistry: New Synthetic Directions, Technologies, Materials, and Biological Applications. ACS Symposium Series No. 949; Soloshonok, V. A.; Mikami, K.; Yamazaki, T.; Welch, J. T.; Honek, J., Eds., American Chemical Society: Washington, DC, 2007; pp 477-486.


Progress 01/01/06 to 12/31/06

Outputs
No progress reported this period

Impacts
Our efforts are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel proteins with desirable properties. These novel proteins could be useful as chemotherapeutic agents or biotechnological tools.

Publications

  • Allen, M.J., Raines, R.T. and Kiessling, L.L. 2006. Contrast agents for magnetic resonance imaging synthesized with ring-opening metathesis polymerization. J. Am. Chem. Soc. 128:6534-6535.
  • Horng, J.-C. and Raines, R.T. 2006. Stereoelectronic effects on polyproline conformation. Protein Sci. 15:74-83.
  • Kotch, F.W. and Raines, R.T. 2006. Self-assembly of synthetic collagen triple helices. Proc. Natl. Acad. Sci. USA 103:3028-3033.
  • Eckenroth, B., Harris, K., Turanov, A.A., Gladyshev, V.N., Raines, R.T. and Hondal, R.J. Semisynthesis and characterization of mammalian thioredoxin reductase. 2006. Biochemistry 45:5158-5170.
  • Lavis, L.D., Chao, T.-Y. and Raines, R.T. 2006. Fluorogenic label for biomolecular imaging. ACS Chem. Biol. 1:252-260.
  • Shoulders, M.D., Hodges, J.A. and Raines, R.T. 2006. Reciprocity of steric and stereoelectronic effects in the collagen triple helix. J. Am. Chem. Soc. 128:8112-8113.
  • Soellner, M.B., Nilsson, B.L. and Raines, R.T. 2006. Reaction mechanism and kinetics of the traceless Staudinger ligation. J. Am. Chem. Soc. 128:8820-8828.
  • Lavis, L.D., Chao, T.Y. and Raines, R.T. 2006. Latent red and blue fluorophores based on the trimethyl lock. ChemBioChem 7:1151-1154.
  • Smith, B.D. and Raines, R.T. 2006. Genetic selection for critical residues in ribonucleases. J. Mol. Biol. 362:459-478.
  • Liu, X., Jang, C.-H., Zheng, F., Jurgensen, A., Denlinger, J.D., Dickson, K.A., Raines, R.T., Abbott, N.L. and Himpsel, F.J. 2006. Characterization of protein immobilization at silver surfaces by near-edge X-ray absorption fine structure spectroscopy. Langmuir 22:7719-7725.
  • Kalia, J. and Raines, R.T. 2006. Reactivity of intein thioesters: Appending a functional group to a protein. ChemBioChem 7:1375-1383.
  • Schlieve, C.R., Tam, A., Nilsson, B.L., Lieven, C.J., Raines, R.T. and Levin, L.A. 2006. Synthesis and characterization of a novel class of reducing agents that are highly neuroprotective for retinal ganglion cells. Exp. Eye Res. 83:1252-1259.
  • Hodges, J.A. and Raines, R.T. 2006. Energetics of an n->pi* interaction that impacts protein structure. Org. Lett. 8:4695-4697.
  • Macrocyclic scaffold for the collagen triple helix. Horng, J.-C., Hawk, A.J., Zhao, Q., Benedict, E.S., Burke, S.D. and Raines, R.T. 2006. Org. Lett. 8:4735-4738.
  • Johnson, R.J., Lin, S.R. and Raines, R.T. 2006. A ribonuclease zymogen activated by the NS3 protease of the hepatitis C virus. FEBS J. 273:5457-5465.
  • Soellner, M.B., Tam, A. and Raines, R.T. 2006. Staudinger ligation of peptides at non-glycyl residues. J. Org. Chem. In Press.
  • Raines, R.T. 2006. 2005 Emil Thomas Kaiser Award. Protein Sci. 15:1219-1225.
  • Fuchs, S.M. and Raines, R.T. 2006. Internalization of cationic peptides: The road less (or more?) traveled.Cell. Mol. Life Sci. 63:1819-1822.
  • Kiessling, L.L., Wedde, S.G. and Raines, R.T. 2006. Symbiosis: Chemical Biology at Wisconsin. ACS Chem. Biol. 1:481-484.


Progress 01/01/05 to 12/31/05

Outputs
The amino acid sequence of a protein encodes its three-dimensional structure, and this structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, my research group continues to illuminate in atomic detail both the chemical basis and the biological purpose for protein structure and protein function.

Impacts
Our efforts are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel proteins with desirable properties.

Publications

  • Synthetic surfaces for nuclease adsorption. Bryan D. Smith, Matthew B. Soellner, and Ronald T. Raines (2005). Langmuir 21, 187-190.
  • O-Acylation of hydroxyproline residues: Effect on peptide-bond isomerization and collagen stability. Cara L. Jenkins, Alexander I. McCloskey, Ilia A. Guzei, Eric S. Eberhardt, and Ronald T. Raines (2005). Peptide Science 80, 1-8.
  • Latent fluorophore based on the trimethyl lock. Sunil S. Chandran, Kimberly A. Dickson, and Ronald T. Raines (2005). Journal of the American Chemical Society 127, 1652-1653.
  • Binding of nonnatural 3?-nucleotides to ribonuclease A. Cara L. Jenkins, Nethaji Thiyagarajan, Rozamond Y. Sweeney, Michael P. Guy, Bradley R. Kelemen, K. Ravi Acharya, and Ronald T. Raines (2005). FEBS Journal 272, 744-755.
  • Polyarginine as a multifunctional fusion tag. Stephen M. Fuchs and Ronald T. Raines (2005). Protein Science 14, 1538-1544.
  • Peptide bond isosteres: Ester or (E)-alkene in the backbone of the collagen triple helix. Cara L. Jenkins, Melissa M. Vasbinder, Scott J. Miller, and Ronald T. Raines (2005). Organic Letters 7, 2619-2622.
  • Reconstitution of a defunct glycolytic pathway via recruitment of ambiguous sugar kinases. Brian G. Miller and Ronald T. Raines (2005). Biochemistry 44, 10776-10783.
  • Catalysis of protein disulfide bond isomerization in a homogeneous substrate. Elizabeth A. Kersteen, Seth R. Barrows, and Ronald T. Raines (2005). Biochemistry 44, 12168-12178.
  • Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A. Thomas J. Rutkoski, Erin L. Kurten, Julie C. Mitchell, and Ronald T. Raines (2005). Journal of Molecular Biology 354, 41-54.
  • Stereoelectronic and steric effects in the collagen triple helix: Toward a code for strand association. Jonathan A. Hodges and Ronald T. Raines (2005). Journal of the American Chemical Society 127, 15923-15932.
  • Cytotoxicity of bovine seminal ribonuclease: Monomer versus dimer. J. Eugene Lee and Ronald T. Raines (2005). Biochemistry 44, 15760-15767.
  • Chemical synthesis of proteins. Bradley L. Nilsson, Matthew B. Soellner, and Ronald T. Raines (2005). Annual Review of Biophysics and Biomolecular Structure 34, 91-118.
  • Ribonuclease inhibitor: Structure and function. Kimberly A. Dickson, Marcia C. Haigis, and Ronald T. Raines (2005). Progress in Nucleic Acid Research and Molecular Biology 80, 349-374.


Progress 01/01/04 to 12/31/04

Outputs
My research group is using the tools of biochemistry, biophysics, chemistry, and molecular biology to illuminate in atomic detail the basis for protein structure and function. The overall goals of our research are (1) to understand how the amino acid sequence of a protein encodes its three-dimensional structure, (2) to understand how this structure manifests itself in function, and (3) to create new proteins having desirable properties. During the last period, we made substantial progress towards achieving all three of these goals.

Impacts
Our efforts are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel proteins with desirable properties.

Publications

  • Glycosylation of onconase increases its conformational stability and toxicity for cancer cells. Byung-Moon Kim, Hana Kim, Ronald T. Raines, and Younghoon Lee (2004) Biochemical and Biophysical Research Communications 315, 976-983.
  • Pathway for polyarginine entry into mammalian cells. Stephen M. Fuchs and Ronald T. Raines (2004). Biochemistry 43, 2438-2444.
  • Identifying latent enzyme activities: Substrate ambiguity within modern bacterial sugar kinases. Brian G. Miller and Ronald T. Raines (2004). Biochemistry 43, 6387-6392.
  • Zinc(II)-mediated inhibition of ribonuclease Sa by an N-hydroxyurea nucleotide and its basis. Alexander A. Makarov, Gennady I. Yakovlev, Vladimir A. Mitkevich, Joshua J. Higgin, and Ronald T. Raines (2004). Biochemical and Biophysical Research Communications 319, 152-156.
  • Imaging the binding ability of proteins immobilized on surfaces with different orientations by using liquid crystals. Yan-Yeung Luk, Matthew L. Tingey, Kimberly A. Dickson, Ronald T. Raines, and Nicholas L. Abbott (2004). Journal of the American Chemical Society 126, 9024-9032.
  • Production of human prolyl 4-hydroxylase in Escherichia coli. Elizabeth A. Kersteen, Joshua J. Higgin, and Ronald T. Raines (2004). Protein Expression & Purification 38, 279-291.
  • Substituted 2-azabicyclo[2.1.1]hexanes as constrained proline analogues: Implications for collagen stability. Cara L. Jenkins, Guoliang Lin, Jingqi Duo, Deepa Rapolu, Ilia A. Guzei, Ronald T. Raines, and Grant R. Krow (2004). Journal of Organic Chemistry 69, 8565-8573.
  • Fluorescence gel retardation assay to detect protein - protein interactions. Sang-Hyun Park and Ronald T. Raines (2004). In Methods in Molecular Biology, vol. 261: Protein - Protein Interactions: Methods and Protocols (Haian Fu, Ed.), pp. 155-160, Humana Press, Totowa, NJ.
  • Fluorescence polarization assay to quantify protein - protein interactions. Sang-Hyun Park and Ronald T. Raines (2004). In Methods in Molecular Biology, vol. 261: Protein - Protein Interactions: Methods and Protocols (Haian Fu, Ed.), pp. 161-165, Humana Press, Totowa, NJ.
  • Active site of ribonuclease A. Ronald T. Raines (2004). In Artificial Nucleases (Marina A. Zenkova, Ed.), pp. 19-32, Springer - Verlag, Heidelberg, Germany.


Progress 01/01/03 to 12/31/03

Outputs
The amino acid sequence of a protein encodes its three-dimensional structure, and that structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, we are illuminating in atomic detail the chemical basis and biological purpose for protein structure and protein function.

Impacts
Our efforts are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel molecules with desirable properties.

Publications

  • Nilsson, B.L., Hondal, R.J., Soellner, M.B. and Raines, R.T. 2003. Protein assembly by orthogonal chemical ligation methods. J. Am. Chem. Soc. 125:5268-5269.
  • Woycechowsky, K.J. and Raines, R.T. 2003. The CXC motif: A functional mimic of protein disulfide isomerase. Biochemistry 42:5387-5394.
  • Hinderaker, M.P. and Raines, R.T. 2003. An electronic effect on protein structure. Protein Sci. 12:1188-1194.
  • Wright, G., Higgin, J.J., Raines, R.T., Steenbergen, C. and Murphy, E. 2003. Activation of the prolyl hydroxylase oxygen-sensor results in induction of GLUT1, HO-1 and NOS-2 proteins in cultured neonatal cardiomyocytes. J. Biol. Chem. 278:20235-20239.
  • Jenkins, C.L., Bretscher, L.E., Guzei, I.A. and Raines, R.T. 2003. Effect of 3-hydroxyproline residues on collagen stability. J. Am. Chem. Soc. 125:6422-6427.
  • Smith, B.D., Soellner, M.B. and Raines, R.T. 2003. Potent inhibition of ribonuclease A by a oligo(vinylsulfonic acid). J. Biol. Chem. 278:20934-20938.
  • Arnold, U., Hinderaker, M.P., Koditz, J., Golbik, R., Ulbrich-Hofmann, R. and Raines, R.T. 2003. Protein prosthesis: A nonnatural residue accelerates folding and increases stability. J. Am. Chem. Soc. 125:7500-7501.
  • Dickson, K.A., Dahlberg, C.L. and Raines, R.T. 2003. Compensating effects on the cytotoxicity of ribonuclease A variants. Arch. Biochem. Biophys. 415:172-177.
  • Woycechowsky, K.J., Hook, B.A. and Raines, R.T. 2003. Catalysis of protein folding by an immobilized small-molecule dithiol. Biotechnol. Prog. 19:1307-1314.
  • Hodges, J.A. and Raines, R.T. 2003. Stereoelectronic effects on collagen stability: The dichotomy of 4-fluoroproline diastereomers. J. Am. Chem. Soc. 125:9262-9263.
  • Soellner, M.B., Dickson, K.A., Nilsson, B.L. and Raines R.T. 2003. Site-specific protein immobilization by Staudinger ligation. J. Am. Chem. Soc. 125:11790-11791.
  • Lee, J.E. and Raines, R.T. 2003. Contribution of active-site residues to the function of onconase, a ribonuclease with antitumoral activity. Biochemistry 42:11443-11450.
  • Matousek, J., Soucek, J., Slavik, T., Tomanek, M., Lee, J.E. and Raines, R.T. 2003. Comprehensive comparison of the cytotoxic activities of onconase and bovine seminal ribonuclease. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 136:In Press.
  • Kersteen, E.A. and Raines, R.T. 2003. Catalysis of disulfide bond formation by protein disulfide isomerase and small-molecule mimics. Antioxid. Redox. Signal. 5:413-424.
  • Dickson K.A., Leland P.A. and Raines, R.T. 2003. Exploring the role of the ribonuclease inhibitor protein in angiogenesis. Biochemistry 42:28.
  • Luk, Y.-Y., Abbott, N.L., Raines, R.T. Tingey, M.L. and Dickson, K.A. 2003. Comparison of the binding activity of randomly oriented and uniformly oriented proteins immobilized by chemoselective coupling to a self-assembled monolayer. Biochemistry 42:247.
  • Haigis, M.C. and Raines, R.T. 2003. Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway. J. Cell. Sci. 116:313-324.
  • Plainkum, P., Fuchs, S.J., Wiyakrutta, S. and Raines, R.T. 2003. Creation of a zymogen. Nat. Struct. Biol. 10:115-119.
  • Haigis, M.C., Kurten, E.L. and Raines, R.T. 2003. Ribonuclease inhibitor as an intracellular sentry. Nucleic Acids Res. 31:1024-1032.
  • Higgin, J.J., Yakovlev, G.I., Mitkevich, V.A., Makarov, A.A. and Raines, R.T. 2003. Zinc(II)-mediated inhibition of a ribonuclease by an N-hydroxyurea nucleotide. Bioorg. Med. Chem. Lett. 13:409-412.
  • Park, C. and Raines, R.T. 2003. Catalysis by ribonuclease A is limited by the rate of substrate association. Biochemistry 42:3509-3518.
  • Nilsson, B.L., Soellner, M.B. and Raines, R.T. 2003. Protein assembly using the Staudinger ligation. Biopolymers 71:P159.
  • Nilsson, B.L., Soellner, M.B. and Raines, R.T. 2003. Protein assembly using the Staudinger ligation. Biopolymers 71:P002.
  • Hodges, J.A. and Raines, R.T. 2003. The effect of fluoroproline in the X-position on the stability of the collagen triple helix. Biopolymers 71:P032.


Progress 01/01/02 to 12/31/02

Outputs
The amino acid sequence of a protein encodes its three-dimensional structure, and this structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, we are illuminating in atomic detail both the chemical basis and the biological purpose for protein structure and protein function.

Impacts
Our efforts are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel molecules with desirable properties.

Publications

  • Umezawa, N., Gelman, M.A., Haigis, M.C., Raines, R.T. and Gellman, S.H. 2002. Translocation of a beta-peptide across cell membranes. J. Am. Chem. Soc. 124:368-369.
  • Leland, P.A., Staniszewski, K.E., Park, C., Kelemen, B.R. and Raines, R.T. 2002. The ribonucleolytic activity of angiogenin. Biochemistry 41:1343-1350.
  • DeRider, M.L., Wilkens, S.J., Waddell, M.J., Bretscher, L.E., Weinhold, F., Raines, R.T. and Markley, J.L. 2002. Collagen stability: Insights from NMR spectroscopic and hybrid density functional computational investigations of the effect of electronegative substituents on prolyl ring conformations. J. Am. Chem. Soc. 124:2497-2505.
  • Haigis, M.C., Kurten, E.L., Abel, R.L. and Raines, R.T. 2002. KFERQ sequence in ribonuclease A-mediated cytotoxicity. J. Biol. Chem. 277:11576-11581.
  • Haigis, M.C., Haag, E.S. and Raines, R.T. 2002. Evolution of ribonuclease inhibitor by exon duplication. Mol. Biol. Evol. 19:960-964.
  • Abel, R.L., Haigis, M.C., Park, C. and Raines, R.T. 2002. Fluorescence assay for the binding of ribonuclease A to the ribonuclease inhibitor protein. Anal. Biochem. 306:100-107.
  • Soellner, M.B., Nilsson, B.L. and Raines, R.T. 2002. Staudinger ligation of alpha-azido acids retains stereochemistry. J. Org. Chem. 67:4993-4996. Arnold, U., Hinderaker, M.P., Nilsson, B.L. Huck, B.R., Gellman, S.H. and
  • Raines, R.T. 2002. Protein prosthesis: A semisynthetic enzyme with a beta-peptide reverse turn. J. Am. Chem. Soc. 124:8522-8523.
  • Sevcik, J., Urbanikova, L., Leland, P.A. and Raines, R.T. 2002. X-Ray structure of two crystalline forms of a Streptomycete ribonuclease with cytotoxic activity. J. Biol. Chem. 277:47325-47330.
  • Jenkins, C.L. and Raines, R.T. 2002. Insights on the conformational stability of collagen. Nat. Prod. Rep. 19:49-59.
  • Hondal, R.J. and Raines, R.T. 2002. Semisynthesis of proteins containing selenocysteine. Methods Enzymol. 347:70-83.
  • Arnold, U., Hinderaker, M.P. and Raines, R.T. 2002. Semisynthesis of protein variants using intein-mediated protein ligation. TheScientificWorldJOURNAL 2:1823-1827.
  • Park, S.-H. and Raines, R.T. 2002. Genetic screen to dissect protein - protein interactions: Ribonuclease inhibitor - ribonuclease A as a model system. Methods 28:346-352.
  • Gellman, S.H. and Raines, R.T. 2002. This year's model. Curr. Opin. Chem. Biol. 6:727-728.


Progress 01/01/01 to 12/31/01

Outputs
The amino acid sequence of a protein encodes its three-dimensional structure, and this structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, we are illuminating in atomic detail both the chemical basis and the biological purpose for protein structure and protein function.

Impacts
Our efforts are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel molecules with desirable properties.

Publications

  • Nilsson, B.L., Kiessling, L.L. and Raines, R.T. 2001. High-yielding Staudinger ligation of a phosphinothioester and azide to form a peptide. Org. Lett. 3:9-12.
  • Bretscher, L.E., Jenkins, C.L., Taylor, K.M., DeRider, M.L. and Raines, R.T. 2001. Conformational stability of collagen relies on a stereoelectronic effect. J. Am. Chem. Soc. 123:777-778.
  • Park, C., Schultz, L.W. and Raines, R.T. 2001.Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding. Biochemistry 40:4949-4956.
  • Kersteen, E.A. and Raines, R.T. 2001. Contribution of tertiary amides to the conformational stability of collagen triple helices. Biopolymers 59:24-28.
  • Hondal, R.J., Nilsson, B.L. and Raines, R.T.. 2001. Selenocysteine in native chemical ligation and expressed protein ligation. J. Am. Chem. Soc. 123:5140-5141.
  • Klink, T.A., Vicentini, A.M., Hofsteenge, J. and Raines. R.T. 2001. High-level soluble production and characterization of porcine ribonuclease inhibitor. Protein Express. Purif. 22:174-179.
  • Jardine, A.M., Leonidas, D.D., Jenkins, J.L., Park, C., Raines, R.T., Acharya, K.R. and Shapiro, R. 2001. Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin. Biochemistry 40:10262-10272.
  • Park, C. and Raines, R.T. 2001. Quantitative analysis of the effect of salt concentration on enzymatic catalysis. J. Am. Chem. Soc. 123:11472-11479.
  • Leland, P.A., Staniszewski, K.E., Kim, B.-M. and Raines, R.T. 2001. Endowing human pancreatic ribonuclease with cytotoxic activity. J. Biol. Chem. 276:43095-43102.
  • Park, C. and Raines, R.T. 2001. Adjacent cysteine residues as a redox switch. Protein Eng. 14:939-942.
  • Leland, P.A. and Raines, R.T. 2001. Cancer chemotherapy--ribonucleases to the rescue. Chem. Biol. 8:4050-413.
  • Park, C., Kelemen, B.R., Klink, T.A., Sweeney, R.Y., Behlke, M.A., Eubanks, S.R. and Raines, R.T. 2001. Fast, facile, hypersensitive assays for ribonucleolytic activity. Methods Enzymol. 341:81-94.


Progress 01/01/00 to 12/31/00

Outputs
The amino acid sequence of a protein encodes its three-dimensional structure, and this structure manifests itself in biological function. Using techniques that range from synthetic chemistry to cell biology, the Raines group is illuminating in atomic detail both the chemical basis and the biological purpose for protein structure and protein function.

Impacts
Our efforts of the Raines group are leading to insights into the relationship between amino acid sequence and protein function (or dysfunction), as well as to the creation of novel molecules with desirable properties.

Publications

  • Dimer formation by a "monomeric" protein. Chiwook Park and Ronald T. Raines (2000). Protein Science 9, 2026-2033.
  • Excavating an active site: The nucleobase specificity of ribonuclease A. Bradley R. Kelemen, L. Wayne Schultz, Rozamond Y. Sweeney, and Ronald T. Raines (2000). Biochemistry 39, 14487-14494.
  • The S-Tag fusion system for protein purification. Ronald T. Raines, Thomas R. Van Oosbree, and Robert C. Mierendorf (2000). Methods in Enzymology 326, 362-376.
  • Green fluorescent protein chimeras to probe protein-protein interactions. Sang-Hyun Park and Ronald T. Raines (2000). Methods in Enzymology 328, 251-261.
  • The stereoelectronic basis of collagen stability. Ronald T. Raines, Lynn E. Bretscher, Steven K. Holmgren, and Kimberly M. Taylor (2000). In Peptides for the New Millennium: Proceedings of the 16th American Peptide Symposium (G. B. Fields, J. P. Tam, and G. Barany, Eds.), pp. 344-346, Kluwer Academic, Boston, MA.
  • Contribution of mainchain-mainchain hydrogen bonds to the conformational stability of triple-helical collagen. Mark A. Danielson and Ronald T. Raines (2000). In Peptides for the New Millennium: Proceedings of the 16th American Peptide Symposium (G. B. Fields, J. P. Tam, and G. Barany, Eds.), pp. 347-348, Kluwer Academic, Boston, MA.
  • Effect of fluoro-substituted proline residues on the conformational stability of triple-helical collagen mimics. Lynn E. Bretscher, Kimberly M. Taylor, and Ronald T. Raines (2000). In Peptides for the New Millennium: Proceedings of the 16th American Peptide Symposium (G. B. Fields, J. P. Tam, and G. Barany, Eds.), pp. 355-356, Kluwer Academic, Boston, MA.
  • Modulating the conformational stability of triple-helical collagen by chemical modification. Cara L. Jenkins, Kimberly M. Taylor, and Ronald T. Raines (2000). In Peptides for the New Millennium: Proceedings of the 16th American Peptide Symposium (G. B. Fields, J. P. Tam, and G. Barany, Eds.), pp. 357-358, Kluwer Academic, Boston, MA.
  • Native disulfide bond formation in proteins. Kenneth J. Woycechowsky and Ronald T. Raines (2000). Current Opinion in Chemical Biology 4, 533-539.
  • Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A. Tony A. Klink, Kenneth J. Woycechowsky, Kimberly M. Taylor, and Ronald T. Raines (2000). European Journal of Biochemistry 267, 566-572.
  • Origin of the "inactivation" of ribonuclease A at low salt concentration. Chiwook Park and Ronald T. Raines (2000). FEBS Letters 468, 199-202.
  • A ribonuclease A variant with low catalytic activity but high cytotoxicity. Lynn E. Bretscher, Richele L. Abel, and Ronald T. Raines (2000). Journal of Biological Chemistry 275, 9893-9896.
  • Prolyl 4-hydroxylase is required for viability and morphogenesis in Caenorhabditis elegans. Lisa Friedman, Joshua J. Higgin, Gary Moulder, Robert Barstead, Ronald T. Raines, and Judith Kimble (2000). Proceedings of the National Academy of Sciences U.S.A. 97, 4736-4741.
  • Sulfur shuffle: Modulating enzymatic activity by thiol-disulfide interchange. June M. Messmore, Steven K. Holmgren, Juneko E. Grilley, and Ronald T. Raines (2000). Bioconjugate Chemistry 11, 408-413.
  • Conformational stability is a determinant of ribonuclease A cytotoxity. Tony A. Klink and Ronald T. Raines (2000). Journal of Biological Chemistry 275, 17463-17467.
  • Staudinger ligation: An amide from a thioester and azide. Bradley L. Nilsson, Laura L. Kiessling, and Ronald T. Raines (2000). Organic Letters 2, 1939-1941.
  • A synapomorphic disulfide bond is critical for the conformational stability and cytotoxicity of an amphibian ribonuclease. Peter A. Leland, Kristine E. Staniszewski, Byung-Moon Kim, and Ronald T. Raines (2000). FEBS Letters 477, 203-207.
  • Genetic selection for dissociative inhibitors of designated protein-protein interactions. Sang-Hyun Park and Ronald T. Raines (2000). Nature Biotechnology 18, 847-851.
  • Decavanadate inhibits catalysis by ribonuclease A. June M. Messmore and Ronald T. Raines (2000). Archives of Biochemistry and Biophysics 381, 25-30.
  • Effect of bovine seminal ribonuclease and its various forms on bovine oocyte maturation. Tomas Slavik, Josef Matousek, Josef Fulka, and Ronald T. Raines (2000). Journal of Experimental Zoology 287, 394-399.
  • Contribution of individual disulfide bonds to the oxidative folding of ribonuclease A. Margherita Ruoppolo, Floriana Vinci, Tony A. Klink, Ronald T. Raines, and Gennaro Marino (2000). Biochemistry 39, 12033-12042.
  • Pentavalent organo-vanadates as transition state analogues for phosphoryl transfer reactions. June M. Messmore and Ronald T. Raines (2000). Journal of the American Chemical Society 122, 9911-9916.
  • A highly active immobilized ribonuclease. Rozamond Y. Sweeney, Bradley R. Kelemen, Kenneth J. Woycechowsky, and Ronald T. Raines (2000). Analytical Biochemistry 286, 312-314.


Progress 01/01/99 to 12/31/99

Outputs
Work on this project (which will now be called "Chemical Biology of Proteins") has been directed towards understanding how the amino acid sequence of a protein confers biological activity.

Impacts
The project has high impact as proteins perform many of the functions necessary for life.

Publications

  • Variants of ribonuclease inhibitor that resist oxidation. Byung-Moon Kim, L. Wayne Schultz, and Ronald T. Raines (1999). Protein Science 8, 430 - 434.
  • A hyperstable collagen mimic. Steven K. Holmgren, Lynn E. Bretscher, Kimberly M. Taylor, and Ronald T. Raines (1999). Chemistry & Biology 6, 63 - 70.
  • His...Asp catalytic dyad of ribonuclease A: Histidine pKa values in the wild-type, D121N, and D121A enzymes. David J. Quirk and Ronald T. Raines (1999). Biophysical Journal 76, 1571 - 1579. Extending the limits to enzymatic catalysis: diffusion of ribonuclease A in one dimension. Bradley R. Kelemen and Ronald T. Raines (1999). Biochemistry 38, 5302 - 5307. Structural changes to ribonuclease A and their effects on biological activity. Josef Soucek, Ronald T. Raines, Monika Haugg, Sun-Ai Raillard-Yoon, and Steven A. Benner (1999). Comparative Biochemistry and Physiology 123C, 103 - 111.
  • Hypersensitive substrate for ribonucleases. Bradley R. Kelemen, Tony A. Klink, Mark A. Behlke, Shad R. Eubanks, Peter A. Leland, and Ronald T. Raines (1999). Nucleic Acids Research 27, 3696 - 3701. (PubMed)
  • Chemical mechanism of DNA cleavage by the homing endonuclease I-PpoI. Stephen J. Mannino, Cara L. Jenkins, and Ronald T. Raines (1999). Biochemistry 38, 16178 - 16186


Progress 01/01/98 to 12/31/98

Outputs
We have shown that the affinity of a ribonuclease for ribonuclease inhibitor protein plays an integral role in defining the potency of a cytotoxic ribonuclease.

Impacts
(N/A)

Publications

  • Ribonucleases endowed with specific toxicity for spermatogenic layers. Josef Matousek, Jan Riha, Josef Soucek, Jin-Soo Kim, Marc Ribo, Peter A. Leland, and Ronald T. Raines (1997). Comparative Biochemistry and Physiology 118B, 881-888.
  • Variants of ribonuclease A with potent cytotoxic activity. Peter A. Leland, L. Wayne Schultz, Byung-Moon Kim, and Ronald T. Raines (1998). Proceedings of the National Academy of Sciences U.S.A., 10407-10412.


Progress 01/01/97 to 12/31/97

Outputs
The overall goal of the proposed research is to reveal how ribonucleases promote tumor cell death. Onconase is the first cancer therapeutic based on the cytotoxicity of ribonucleases. Onconase is a homolog of the well-characterized enzyme ribonuclease A (RNase A), which is not cytotoxic. Their three-dimensional structures diverge in two significant ways: the site in RNase A that binds to the ribonuclease inhibitor protein (RI) endogenous to mammalian cells differs from the analogous region in onconase, and RNase A has an anionic patch absent in onconase. The working hypothesis of this proposal is that these two differences are necessary for the cytotoxic activity of onconase. This hypothesis will be tested by using RNase A as a template for the construction of hybrid proteins. Because RNase A lacks cytotoxic activity, any such activity in a hybrid protein would reveal a biochemical property necessary for cytotoxicity. The specific aims of the proposed research are to determine (1) role of RI binding-by disrupting the RI binding site of RNase A, (2) role of surface charge-by eliminating the anionic patch on RNase A, (3) intracellular routing-by attaching a KDEL tail or fluorescent probe to RNase A to discern the path to cellular RNA, and (4) intracellular RI levels-by using immobilized RNase A to assess the level of metabolically radiolabeled RI in different cell types. Relevant properties of each mutant RNase A is being assessed in comparison to onconase (positive control) and wild-type RNase A (negative control). These properties include cytotoxicity, ribonucleolytic activity, RI binding, pI, and thermal stability. Appropriate mutations will be combined with the goal of enhancing cytotoxicity. Finally, the three-dimensional structures of mutant RNase As with notable cytotoxicity will be determined by X-ray diffraction analysis. The proposed research uses techniques and ideas from biochemistry, biophysics, molecular biology, and cell biology to reveal new insights into the basis of ribonuclease cytotoxicity, and could lead to new ribonucleases for cancer therapy.

Impacts
(N/A)

Publications

  • Ribonucleases endowed with specific toxicity for spermatogenic layers. Matousek J, Riha J, Soucek J, Kim J-S, Ribo M, Leland PA, Raines RT. 1997. Comp Biochem Physiol 118B:881-8.