Development of new NMR techniques and the structure of the N-terminal domain of Escherichia coli DnaB helicas

Sammanfattning: New techniques applicable in biomolecular nuclear magnetic resonance (NMR) spectroscopy were developed. The three-dimensional structure of the N-terminal domain of Escherichia coli DnaB helicase was determined at atomic resolution using heteronuclear multidimensional NMR techniques. Homonuclear semi-selective decoupling during acquisition improves the resolution of an NMR experiment and presents a convenient way of suppressing zero-quantum cross peaks in NOESY and ROESY experiments. The decoupling pulses can, however, result in decoupling sidebands due to the modulation of the effective magnetic field by the decoupling pulses. Two methods for the suppression of the decoupling sidebands were devised. Spin-state selective filters for the separation of doublet components into different subspectra were developed. The filters are versatile building blocks for NMR pulse sequences and can be used in a straightforward manner for the measurement of one-bond coupling constants and relaxation interference effects as well as for the design of TROSY type experiments. A single scan, sensitivity enhanced TROSY experiment with pulsed field gradients for coherence order selection was developed. Since the modified TROSY pulse sequence requires only a single scan (two FIDs per complex data point) it represents a sequence element suitable for inclusion in more complex NMR experiments. DnaB helicase is the primary replicatory helicase in Escherichia coli. Native DnaB is a hexamer of identical 52.3 kDa subunits. Each subunit is comprised of two domains. The structurally well-defined core of the N-terminal domain of DnaB was determined by NMR to comprise residues 24 to 136 by resonance assignments of the flexible residues in two DnaB fragments of different length, DnaB(1-142) and DnaB(1-161). A third construct, DnaB(24-136), was subsequently overexpressed and isotopically enriched for structure determination. The three-dimensional structure was calculated using the program DYANA and energy minimised using the program OPAL. The final 20 conformers had a root mean square deviation relative to the mean structure of 0.51 A for the backbone heavy atoms of residues 29-134. An estimate of the rotational correlation time of DnaB(24-136) and ultracentrifugation data indicated partial self-aggregation of DnaB(24-136) into dimers. Chemical shift changes of amide (15N-1H) groups were measured as a function of protein concentration and identified the dimerisation interface. The dissociation constant was estimated to be in the mM range. A model of the dimer was built based on the identification of the interaction surface and intermolecular nuclear Overhauser effects (NOEs) and is in agreement with residual dipolar couplings. An association-dissociation equilibrium for the N-terminal domain of DnaB has been proposed earlier from electron microscopy data and is thought to be of importance in the functional DnaB hexamer. Interestingly, DnaB(24-136) is structurally related to the primary dimerisation domain of Escherichia coli gyrase A, which may indicate functional similarities.