Antibodies serve important functions in basic and applied biomedical research, diagnostic tests for health care, and in cancer therapy. Many such applications require antibody engineering to improve the affinity and/or selectivity of the antibodies. Such antibody engineering studies will benefit from guidance provided by thorough site-directed mutagenesis experiments performed on a model system that is well characterized structurally. The purpose of the proposed studies is to understand the effect of a wide variety of site-directed mutations on the antigen-binding affinity and selectivity of an antibody that binds sequence-specifically to DNA. The proposed studies will also provide important information about molecular recognition in general and sequence-specific DNA recognition in particular. Inspection of the uncomplexed and antigen complexed structures of an anti- DNA antibody suggests a wide variety of site-directed mutations that will directly impact: Interdomain protein-protein interactions, the flexibility of an antigen binding loop, and electrostatic interactions with DNA.
The specific aims of the proposed studies are to 1) Analyze existing experimental data for the purpose of understanding antibody sequence and structural data at these sites, 2) Perform detailed molecular modeling of the site-directed molecular modeling of the site-directed mutations, and 3) Make biochemical measurements of the antigen binding affinities and selectivities of the site-directed mutant proteins.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA077636-04
Application #
6475811
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Gallahan, Daniel L
Project Start
1998-12-21
Project End
2003-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
4
Fiscal Year
2002
Total Cost
$105,733
Indirect Cost
Name
University of Vermont & St Agric College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Murphy, Joan A; Barrantes-Reynolds, Ramiro; Kocherlakota, Rama et al. (2004) The CDKN2A database: Integrating allelic variants with evolution, structure, function, and disease association. Hum Mutat 24:296-304