Molecular mechanisms of gene regulation studied by site-directed spin labeling.

摘要

The technique of site-directed spin labeling using cysteine substitution mutagenesis followed by modification of the sulfhydryl group with a nitroxide reagent is emerging as a valuable alternative for the determination of protein folds and conformational changes in a variety of systems. The incorporation of pairs of nitroxides allows determination of intramolecular distances and distance changes with a spatial resolution at the level of the backbone fold under conditions relevant to function. The methodology of electron paramagnetic resonance spectral data acquisition and interpretation is reviewed with studies on conformational changes of Tet repressor (TetR) and the human immunodeficiency virus type 1 reverse transcriptase (RT) on interaction with nucleic acid substrates or inhibitors in solution. A twisting motion of the DNA reading heads of TetR on induction by tetracycline (tc) is observed in solution by changes of the interspin distances between interacting nitroxides at positions 22/22(') or 47/47('). Spin-label side chains located near the tc-binding pocket or at position 202 indicate different conformations for the tc- and DNA-complexed repressor also in the core of the protein. Interspin distances between spin-labeled residue positions 24 and 287 in the fingers and the thumb domains of RT complexed with dsDNA or a pseudoknot RNA in solution were found to agree with the respective crystal data of the so-called open and closed conformations. For the unliganded RT a temperature-dependent equilibrium between these two states is observed.