Many methods are used for screening/characterisation of strong protein-protein interactions (PPIs), but there has been slow progress in understanding weak PPIs due to technical difficulties. In this thesis, combined biophysical and structural approaches were used to study weak PPIs in a model cytochrome P450 (P450) system. P450s are biotechnologically important heme-binding oxidase enzymes involved in lipid, steroid and drug metabolism. Bacillus megaterium P450 BM3 (BM3) was used as a model. BM3 is a high activity P450 with its redox partner (an FAD- and FMN-binding reductase) linked to the P450 in a single polypeptide. Genetic dissection enabled generation of individual P450 (BMP) and P450 reductase (BMR) domains, as well as separate FAD- and FMN-binding domains. Preliminary work on BM3, BMP and BMR used equilibrium optical binding and showed blue (type I) heme Soret band shifts with substrates (N-palmitoylglycine, lauric acid), and red (type II) shifts with inhibitors (imidazole, 4-phenyl imidazole), consistent with typical P450 behaviour. Steady-state kinetic catalytic efficiency of BM3 in reducing cytochrome c was higher than for BMR, suggesting that BMP promotes interactions between BMR/cytochrome c. Interactions between BMP/BMR domains were studied by examining NADPH-dependent electron transfer (e.t.) rate between these domains using stopped-flow absorption spectroscopy, and across a range of ionic strengths. The inter-domain e.t. rate in the reconstituted system was ~1000-fold less than in intact BM3. Elevated ionic strength decreased the e.t. rate in both systems, and particularly for the reconstituted domains. Slow BMP reduction by NADPH in absence of BMR was also observed over longer time scales.Biophysical methods were used in quaternary structural characterization of BM3 and its domains. BM3 reversibly dimerizes (mostly stabilized by electrostatic interactions) with a dissociation constant (Kd) of 0.29 µM. Reversible dimerization was also seen for BMR, but with a weaker Kd than BM3. The FAD domain is near-fully dimerized most likely due to intermolecular disulphide bonds, while FMN and heme domains are monomers. Results indicate that BMR domain interactions regulate BM3 dimerization. The BM3 shape is compact and globular, BMR is compact and ellipsoidal, and the FAD domain is globular with extended ends. Differences between FAD domain crystal and solution structures suggest conformational differences possibly due to disulphide bonds stabilizing solution dimer. 6 different BMR conformations were obtained by rigid body modelling, all showing that BMR exists in an "open" conformation with respect to orientation of its FAD and FMN domains.
|Date of Award||1 Aug 2014|
- The University of Manchester
|Supervisor||Robin Curtis (Supervisor) & Andrew Munro (Supervisor)|