The focus of this proposal is to develop automated design techniques for the structure-based design and engineering of function in proteins. We will accomplish this by the introduction of new ligand-binding sites into proteins of known structure, and the redesign of the properties of existing ligand-binding sites. We propose to extend our previous work in which we have successfully used a structure-based computational approach, the DEZYMER program, to build metal centers with a variety of different activities in the hydrophobic core of a model protein. Here we propose to develop novel computational techniques that introduce further important structural degrees of freedom into the design algorithm. We also propose to develop novel algorithms for the structure-based redesign of ligand binding sites. This procedures will be tested experimentally by optimizing the stabilities and reactivities of a series of rationally designed metal centers; by structure-based design of metalloenzyme active sites in several different protein scaffolds, including thioredoxin, fluorescent green protein and fatty acid binding protein; and by the structure-based redesign of the specificity of a bacterial receptor protein. Central to this approach is the design cycle in which modeling and experimental analysis alternate, thereby iteratively testing and improving both the designs and the computational methods. We propose to extend this approach by introducing a directed evolution stage to improve the designs by experimental selection and screening methods. The development of methodologies for structure-based redesign of protein structure and function have important industrial and biomedical applications such as the development of new catalysts, biosensors, novel biomaterials, and protein-based pharmaceuticals.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049871-07
Application #
6180364
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Jones, Warren
Project Start
1994-04-01
Project End
2003-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
7
Fiscal Year
2000
Total Cost
$222,501
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705