The objective of this program is to understand specific protein-DNA recognition and binding dynamics in solution. NMR spectroscopy and genetic engineering methods are being used to characterize structural and functional features of the interaction. The biological system under investigation is the methionine aporepressor protein dimer, metJ, its corepressor S-adenosylmethionine, and synthetic fragments of the met operator DNA. Based on recent crystallographic data, this repressor protein appears to represent a new DNA binding motif involving insertion of two antiparallel strands, one from each protein monomer, into the major groove of the duplex, and provides a complex but tractable system for detailed NMR investigation. The overall goal is a better understanding of how these interactions control the expression of genes in the methionine regulon. To achieve these objectives, heteronuclear and homonuclear NMR methods are being used along with spectral editing techniques on protein uniformly labeled with C-13 and N-15 isotopes. Using specific resonance assignments in both the protein and in operator fragments of varying length and position of the consensus sequence DNA, the contact regions in the bound complex with and without S-adenosylmethionine are being probed via protein-DNA titration studies monitored by 1D spectroscopy and both 1D and 2D nuclear Overhauser effect spectroscopy. Use of spectral editing techniques with the labeled protein bound to unlabeled DNA and SAM is planned to explore configurations of the bound protein, DNA, and SAM in solution. The DNA binding characteristics of selected mutant proteins containing single amino acid substitutions will also be studied based on results of gel shift and DNase protection screening. With the crystallographic representation as a starting point, dynamical features of this nev binding motif will be systematically examined in solution.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041829-06
Application #
2181087
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1989-04-01
Project End
1996-06-30
Budget Start
1994-12-01
Budget End
1996-06-30
Support Year
6
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Duke University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705