Development of new NMR techniques and structural characterization of complexes between the N-terminal domain of the E. coli arginine repressor and operator DNA

Abstract

This thesis includes the development of new solution NMR methods for the study of biomolecules and the characterization of complexes between the N-terminal domain of the E. coli arginine repressor (ArgR-N) and operator DNA. [alpha]/[beta]-half-filter were designed as spin-state selective pulse sequence elements for the rapid measurement of one-bond coupling constants. The spectral editing of IS cross-peak multiplets into separate subspectra retains the sensitivity of the parent experiment recorded without decoupling during acquisition while maintaining the resolution of the decoupled experiment. Incorporation of the filters into HSQC- and HMQC-type experiments with and without sensitivity enhancement is demonstrated. The original TROSY experiment was re-examined and a generalized version presented, which allows the spin-state selective editing of the multiplet components of 1H-15N groups into four different subspectra without any loss of sensitivity, A gain of [IMAGE] in sensitivity is possible if only two of the four components are selected. The time-shared X([omega]1)-half-filter reduces the loss of magnetization by relaxation by utilizing the filter delay for simultaneous chemical shift evolution. A timeshared 13C([omega]1)-half-filtered 1H NOESY of a 2:1 complex between 13 C-labelled ArgR-N and 16-mer operator DNA showed an increased signal intensity of 30% over the conventional filter. The full-length E. coli arginine repressor (ArgR) binds to DNA as a hexamer. DNA-binding is mediated by pairs of N-terminal domains of ArgR (ArgR-N) which bind to largely palindromic sites, referred to as ARG-boxes, on the operator DNA. NMR data collected for the 2:1 complex between ArgR-N and 16-mer operator DNA include chemical shift perturbation, amide 1H protection from solvent and a relaxation enhancing agent in the solution, relaxation enhancement by spin-labelled DNA and residual dipolar couplings, The interaction surface of the protein was found to include parts of the N-terminal half of helix 3 and the preceding loop, the N-terminal of helix 1, the flexible N-terminus and the sidechain of Gln26. The orientation of the ArgR-N molecules in the 2:1 complex brings the [beta]-fingers in spatial proximity near the centre of the ARG-box. A model of the 2:1 complex is proposed which accounts for most of the NMR and available biochemical data.