Objective The purpose of this project is to devise means and methods to exert control over the rate of electron transfer between two redox centres embedded in an engineered protein matrix. The technological aim is the rational design of redox proteins with desired properties, with the long term objective of achieving catalytic activity for clean technology. This will be achieved by design and construction of chimeric proteins by fusion of small and well characterised electron transferases. The possibility of using chimeras where the electron transferase is used as a gate way to transfer electrons to a catalytic subunit is a new fascinating multi-disciplinary field where biocatalysis, fermentation, genetic engineering, molecular spectroscopy and 3D structure determination are essential and complement each other in rational molecular design. The protein matrix containing the two centres will be constructed by linking two (small) redox proteins, 1) by using a non-covalent linker that connects the two redox centres, or 2) by a covalent (genetically engineered) linker, or 3) by a combination of the two techniques. This genetic engineering approach presents a key advantage over the traditional organic synthesis because it uses the natural protein scaffolding to host the redox centres in their optimal micro-environments for activity. Candidate proteins are azurin from P.aeruginosa and A.denitrificans amicyanin and cytochrome-c550 from T. versutus, cytochrome-c553 and flavodoxin from D. wlgaris. These proteins and their mutants will be used in different combinations for the construction of chimeras. The genes and appropriate modification and expression systems for these proteins are available at the participating institutions. The 3D structures of these proteins and many of their mutants are available and this constitutes a good starting advantage in molecular design. A test case for directional electron transfer via an electron transferase gate way is offered by phenol hydroxylase from Acinetobacter radioresistens, a fast phenol and benzoate metabolising strain. This enzyme performs the first step of aromatic biodegradation, the hydroxylation reaction and it contains flavin, iron-sulphur and iron redox centres. Structural and genetic characterization of this enzyme will provide the background for genetic design of a flavodoxin-like driven electron transfer gate way. The structure of this enzyme and of the engineered proteins will be solved by X-ray crystallography. Functional characterization will be achieved with complementary techniques offered across the consortium where state-of-the-art instrumentation is available, ranging from NMR (200-400-600 MHz), EPR, ENDOR, ESEEM, Mossbauer spectroscopy, stopped flow and electrochemistry (square wave voltammetry, cyclic voltammetry and chronoamperometry). The time course of electron transfer will be measured by spectroscopic means such as line broadening effects using NMR and EPR, as well as by direct electrochemistry. Furthermore, micro-spectrophotometry on crystals will allow the determination of whether the proteins retain, in the crystalline state, the conformation which is active in solution. The structural and functional information learnt during the development of this project will guide further engineering efforts in the production of functional and novel redox proteins with the desired properties. Fields of science natural scienceschemical scienceselectrochemistrynatural sciencesearth and related environmental sciencesgeologymineralogycrystallographymedical and health sciencesmedical biotechnologygenetic engineeringnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymesnatural sciencesphysical sciencesopticsspectroscopy Programme(s) FP4-BIOTECH 2 - Specific research, technological development and demonstration programme in the field of biotechnology, 1994-1998 Topic(s) 0601 - Structure-function relationships Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE EU contribution No data Address SW7 2AY London United Kingdom See on map Total cost No data Participants (5) Sort alphabetically Sort by EU Contribution Expand all Collapse all LEIDEN UNIVERSITY Netherlands EU contribution No data Address 55,Einsteinweg 55 Gorlaeus Laboratoria 2300 RA Leiden See on map Total cost No data MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V. Germany EU contribution No data Address 18A,Am Klopferspitz 18A 82152 PLANEGG / KRAILLING See on map Total cost No data Universidade Nova de Lisboa Portugal EU contribution No data Address 6,Quinta do Marqués 2780 Oeiras See on map Total cost No data Università degli Studi di Parma Italy EU contribution No data Address Viale delle Scienze 78 43100 Parma See on map Total cost No data Università degli Studi di Torino Italy EU contribution No data Address Via Accademia Albertina 17-34 10123 Torino See on map Total cost No data
