Quantitative determination of conformational, dynamic, and kinetic parameters of a ligand-protein/DNA complex from a complete relaxation and conformational exchange matrix analysis of intermolecular transferred NOESY.
We report a quantitative analysis of the 13C-edited intermolecular transferred NOESY (inter-TrNOESY) spectrum of the trp-repressor/operator complex (trp-rep/op) with [ul-13C/15N]-L-tryptophan corepressor using a computer program implementing complete relaxation and conformational exchange matrix (CORCEMA) methodology [Moseley et al. (1995) J. Magn. Reson. 108B, 243-261]. Using complete mixing time curves of three inter-TrNOESY peaks between the tryptophan and the Trp-rep/op, this self-consistent analysis determined the correlation time of the bound species (tauB = 13.5 ns) and the exchange off-rate (k(off) = 3.6 s(-1)) of the corepressor. In addition, the analysis estimated the correlation time of the free species (tauF approximately 0.15 ns). Also, we demonstrate the sensitivity of these inter-TrNOESY peaks to several factors including the k(off) and orientation of the tryptophan corepressor within the binding site. The analysis indicates that the crystal structure orientation for the corepressor is compatible with the solution NMR data.
Moseley
H N
HN
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294-2041, USA.
Lee
W
W
Arrowsmith
C H
CH
Krishna
N R
NR
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins