Enzymes and structural proteins play important roles in human physiology as well as hold a potential usefulness in industrial and clinical applications, such as bioremediation and therapeutic drug design. Understanding how protein structure relates to function allows us to determine the role of abnormal proteins in disease, and is necessary for the success of protein engineering. The main goal of this proposal is to use a new combinatorial form of random mutagenesis or """"""""directed evolution"""""""" called DNA shuffling to study how mutations in all regions of protein structure affect the specificity of ligand binding and catalysis. Rather than starting with preconceived hypotheses of what structural features of a protein affect these characteristics, this method allows nature to provide us with the solutions. Isocitrate dehydrogenase (IDH) will be randomly mutagenized and mutants that exhibit altered substrate specificity and can use isopropylmalate as a substrate will be selected using a bacterial growth assay. Because IDH is well characterized structurally and mechanistically, detailed analyses of the mutants by kinetic studies and crystallography will be possible. This will provide valuable insights into the structural basis of substrate specificity, and aid in future studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM019582-01
Application #
2708579
Study Section
Special Emphasis Panel (ZRG3-BIO (01))
Project Start
1999-01-12
Project End
Budget Start
1998-09-01
Budget End
1999-08-31
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704