ISSN:
1573-5001
Keywords:
deuterium labelling
;
protein–DNA interactions
;
protein–protein interactions
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of 2H,15N,13C labelled intact and (des2–7) trp repressor (Δ2–7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone 1HN, 15N, 13Cα positions in both forms of the protein and for13 Cβ in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of Δ2–7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178–182]. Comparison of the backbone chemical shifts of DNA-bound Δ2–7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2–12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of Cα chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and Δ2–7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1008257803130
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