NMR assignments for the amino-terminal residues of trp repressor and their role in DNA binding.
The trp repressor of Escherichia coli specifically binds to operator DNAs in three operons involved in tryptophan metabolism. The NMR spectra of repressor and a chymotryptic fragment lacking the six amino-terminal residues are compared. Two-dimensional J-correlated spectra of the two forms of the protein are superimposable except for cross-peaks that are associated with the N-terminal region. The chemical shifts and relaxation behavior of the N-terminal resonances suggest mobile "arms". Spin-echo experiments on a ternary complex of repressor with L-tryptophan and operator DNA indicate that the termini are also disordered in the complex, although removal of the arms reduces the DNA binding energy. Relaxation measurements on the armless protein show increased mobility for several residues, probably due to helix fraying in the newly exposed N-terminal region. DNA binding by the armless protein does not reduce the mobility of these residues. Thus, it appears that the arms serve to stabilize the N-terminal helix but that this structural role does not explain their contribution to the DNA binding energy. These results suggest that the promiscuous DNA binding by the arms seen in the X-ray crystal structure is found in solution as well.
Arrowsmith
C H
CH
Stanford Magnetic Resonance Laboratory, Stanford University Medical Center, California 94305.
Carey
J
J
Treat-Clemons
L
L
Jardetzky
O
O
D001426:Bacterial Proteins; D002918:Chymotrypsin; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009876:Operon; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D002918:Chymotrypsin; D004269:DNA, Bacterial; D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins