J Mol Biol 1999 Jul 2;290(1):229-40

Domain motions accompanying Tet repressor induction defined by
changes of interspin distances at selectively labeled sites.

Tiebel B, Radzwill N, Aung-Hilbrich LM, Helbl V, Steinhoff HJ, Hillen W

Lehrstuhl fur Mikrobiologie Biochemie und, Genetik der Friedrich-Alexander Universitat Erlangen-Nurnberg,
Staudtstrasse 5, Erlangen, 91058, FRG, Germany.

To investigate internal movements in Tet repressor (TetR) during induction by tetracycline (tc) we determined the
interspin distances between pairs of nitroxide spin labels attached to specific sites by electron paramagnetic resonance
(EPR) spectroscopy. For this purpose, we constructed six TetR variants with engineered cysteine pairs located in
regions with presumed conformational changes. These are I22C and N47C in the DNA reading head,
T152C/Q175C, A161C/Q175C and R128C/D180C near the tc-binding pocket, and T202C in the dimerization
surface. All TetR mutants show wild-type activities in vivo and in vitro. The binding of tc results in a considerable
decrease of the distance between the nitroxide groups attached to both I22C residues in the TetR dimer and an
increase of the distance between the N47C residues. These opposite effects are consistent with a twisting motion of
the DNA reading heads. Changes of the spin-spin interactions between nitroxide groups attached to residues near the
tc-binding pocket demonstrate that the C-terminal end of alpha-helix 9 moves away from the protein core upon DNA
binding. Alterations of the dipolar interaction between nitroxide groups at T202C indicate different conformations for
tc and DNA-bound repressor also in the dimerization area. These results are used to model structural changes of
TetR upon induction. Copyright 1999 Academic Press.