This proposal aims at providing basic knowledge relevant to metalloprotein design, the pre-determined placement and alteration of metal centers in proteins. A primary goal is to define the structural basis for the binding and activity of biologically important metal ions in metalloproteins. First, I will use existing metalloprotein structures as templates for the design of metal binding mutants in green fluorescent protein (GFP). These sites will be positioned to shift GGFP fluorescence upon metal ion binding. Successful metal ion binding GFP mutants will be characterized biochemically and crystallographically. Second, I will test the impact of the protein neighborhood by random mutagenesis of sites neighboring the bound metal ion. These random mutants will be selected using the power of cell sorting by fluorescent shifts mediated by metal ion binding to GFP. Third, successful designs will be tested using fluorescent microscopy as possible probes of specific metal ions within eukaryotic cells. Overall, this work uses the synergistic techniques of molecular biology, x-ray crystallography, and cell biology to examine metal ion binding specificity and affinity by proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM019290-01
Application #
2521186
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1998-06-10
Project End
Budget Start
1998-06-01
Budget End
1999-05-31
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037