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

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 var iants with engineered cysteine pairs located in regions with presumed confo rmational changes. These are I22C and N47C in the DNA reading head, T152C/Q 175C, A161C/Q175C and R128C/D180C near the tc-binding pocket, and T202C in the dimerization surface. All TetR mutants show wild-type activities in viv o and in vitro. The binding of t 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. Thes e opposite effects are consistent with a twisting motion of the DNA reading heads. Changes of the spin-spin interactions between nitroxide groups atta ched 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. Al terations of the dipolar interaction between nitroxide groups at T202C indi cate different conformations for re and DNA-bound repressor also in the dim erization area. These results are used to model structural changes of TetR upon induction. (C) 1999 Academic Press.