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 pr operties. The molecular 'Lego' mimics the natural molecular evolution that proceeded by modular assembly of genes/DNA segments. Non-physiological elec tron transfer partners, flavodoxin (fld) and cytochrome c(553) (c(553)) fro m Desulfovibrio vulgaris and the haem domain of P450 BM3 (BMP) from Bacillu s megaterium have been used as building blocks in different combinations to build artificial redox chains. The kinetic characterisation of the electro n transfer (ET) between the separate building blocks has been carried out. Under pseudo-first order conditions, a limiting ET rate, k(lim), of 0.48 +/ -0.05 s(-1) and 43.77 +/-2.18 s(-1) and an apparent binding constant, K-app , of 21 +/-6 muM and 1.23 +/-0.32 muM have been found for the fld/c(553) an d fld/BMP redox pairs, respectively. These results show that fld can be use d as a module for transferring electrons to c(553) and BMP. A 3D model of t he fld/c(553) and fld/BMP complexes was used to guide the construction of c ovalently linked assemblies via engineered disulfide bridges or by fusion o f the relevant genes via an engineered loop. The first approach led to the construction, expression and characterisation of the S35C and S64C mutants of fld and M23C and G51C mutants of c(553). Although the redox potentials o f the separate mutants were found to be the same as those of recombinant wi ld type proteins (-408 mV for the semiquinone/hydroquinone couple of fld an d +32 mV for the c(553)), the c(553) homo-dimers M23C-M23C and G51C-G51C we re found to have redox potentials of +88 and +105 mV, respectively. These d ifferences have been analysed in terms of exposure of the haem cofactors to the solvent, and these lead to some interesting questions on the redox pot entials of the transient redox complexes in physiological systems. The fld- c(553) S64C-M23C and S35C-M23C chimeras were constructed, expressed and pur ified 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 li nked assemblies of both fld-c(553) and fld-BMP. The former was expressed us ing a seven amino acid (GPGPGPG) loop linking the C-terminus of fld to the N-terminus of c(553). The fld-BMP fusion protein was successfully expressed by using the naturally occurring loop of the P450 BM3 (residues 471-479) t o 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 f rom 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 demonstr ates the feasibility of the molecular 'Lego' approach in generating functio nal multi-domain proteins with designed properties, beyond the restrictions imposed by the naturally occurring protein domains.