Engineering artificial redox chains by molecular 'Lego'

This work reports on a novel approach for building artificial redox chains: the molecular 'Lego' approach. This exploits the scaffold of natural redox proteins by fusing together functional protein modules with the desired properties. The molecular 'Lego' mimics the natural molecular evolution that proceeded by modular assembly of genes/DNA segments. Non-physiological electron transfer partners, flavodoxin (fld) and cytochrome c553 (c553) from Desulfovibrio vulgaris and the haem domain of P450 BM3 (BMP) from Bacillus megaterium have been used as building blocks in different combinations to build artificial redox chains. The kinetic characterization of the electron transfer (ET) between the separate building blocks has been carried out. Under pseudo-first order conditions, a limiting ET rate, klim, of 0.48 +/- 0.05 s-1 and 43.77 +/- 2.18 s-1 and an apparent binding constant, Kapp, of 21 +/- 6 microM and 1.23 +/- 0.32 microM have been found for the fld/c553 and fld/BMP redox pairs, respectively. These results show that fld can be used as a module for transferring electrons to c553 and BMP. A 3D model of the fld/c553 and fld/BMP complexes was used to guide the construction of covalently linked assemblies via engineered disulfide bridges or by fusion of the relevant genes via an engineered loop. The first approach led to the construction, expression and characterization of the S35C and S64C mutants of fld and M23C and G51C mutants of c553. Although the redox potentials of the separate mutants were found to be the same as those of recombinant wild type proteins (-408 mV for the semiquinone/hydroquinone couple of fld and +32 mV for the c553), the c553 homo-dimers M23C-M23C and G51C-G51C were found to have redox potentials of +88 and +105 mV, respectively. These differences have been analysed in terms of exposure of the haem cofactors to the solvent, and these lead to some interesting questions on the redox potentials of the transient redox complexes in physiological systems. The fld-c553 S64C-M23C and S35C-M23C chimeras were constructed, expressed and purified but the FMN was found to be destabilised resulting in the apo-form of these proteins. The gene fusion strategy was used to produce covalently linked assemblies of both fld-c553 and fld-BMP. The former was expressed using a seven amino acid (GPGPGPG) loop linking the C-terminus of fld to the N-terminus of c553. The fld-BMP fusion protein was successfully expressed by using the naturally occurring loop of the P450 BM3 (residues 471-479) to link the BMP domain at the N-terminus with fld domain at the C-terminus. This fusion was found to be correctly folded and functional. Efficient ET from the FMN to the haem domain (370 s-1) was also found to be in the same region of the physiological redox partners (250 s-1). This work demonstrates the feasibility of the molecular 'Lego' approach in generating functional multi-domain proteins with designed properties, beyond the restrictions imposed by the naturally occurring protein domains.