A biosynthetic method has been developed that makes possible the site- specific incorporation of a large number of noncoded amino acids and analogues within proteins. In this approach, an amber suppressor tRNA chemically aminoacylated with the desired amino acid incorporates this amino acid site specifically into a protein in response to an amber codon introduced at the corresponding position in the protein's DNA sequence. Using this method, precisely tailored changes within a protein can be made to address specific structure-function questions, including changes in the size, acidity, nucleophility or hydrophobic properties of an amino acid . In addition, analogues with altogether new properties can be introduced including spin labels, photoactivable and redox active amino acids. We will apply this methodology to a series of specific problems associated with protein stability/folding and catalysis. These include the role of hydrogen bonding in stabilizing protein structure, low barrier hydrogen bonds in enzymic catalysis, the GTPase mechanism of small signal transduction proteins (Ras), mechanistic studies of the novel Claisen rearrangement catalyzed by the E. coli and B. substilis chorismate mutases, and the novel radical mechanism of ribonucleotide reductase. We have chosen these systems by virtue of their significance and the fact that noncoded amino acids may provide certain advantages in their study. Finally we also propose to continue to improve the methodology itself.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM049220-05
Application #
2022725
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1993-05-01
Project End
2001-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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