Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.
We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.
Kintrup
M
M
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
Schubert
P
P
Kunz
M
M
Chabbert
M
M
Alberti
P
P
Bombarda
E
E
Schneider
S
S
Hillen
W
W
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry; D013754:Tetracyclines / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; C016229:4-epianhydrotetracycline; D014364:Tryptophan