Electrochemical Functional Imaging of Multi-Component Enzymatic Systems Organized on Virus Nano-Scaffolds – eVIRZYM

The major scientific goal of this project is to generate new insights into the way spatial organization modulates the efficiency of scaffolded multi-component enzymatic systems. To achieve this goal, experimental nanoscale enzymatic platforms need to be designed and their functional behavior interrogated at the individual nano-system scale. Beyond the formidable instrumental challenge this represents, being able to carry out such nanoscale catalytic measurements potentially enables revealing new molecular behaviors inaccessible to “ensemble measurements” classically used in enzymology.
As an original nano-engineering approach for reconstituting functional multi-component enzymatic systems we propose to use bacteriophages (viruses from bacteria) as scaffolds. These virus nano-carriers will be used as intermediate building blocks carrying correctly exposed proteins on their surface, and immobilized onto a solid substrate. Thanks to the expertise of the biologist Partner of the project (Partner 2) in virus and protein engineering a large toolbox of site-selective bio-conjugation strategies, some of them entirely original, will be available for organizing molecular components onto the virus scaffold. Two types of particularly relevant bio-mimetic organized nanosystems will be assembled on viruses: - the first one will incorporate an electron transport chain and a redox enzyme, mimicking a simplified signal transducer of the kind found in cellular mitochondria and plant chloroplasts where electrons are transported between redox enzymes by membrane-confined redox species -the second one will couple a lipase and a redox enzyme assembled in a cascade configuration, an ubiquitous arrangement in natural scaffolded enzymatic systems.
Functional interrogation of the engineered multi-component systems will be carried out using a high resolution correlative local probe microscopy technique combining atomic force (AFM) and electrochemical microscopy (SECM) in a configuration invented by the electrochemist Partner of this project (Partner 1), and uniquely enabling to: (i) resolve the position of the virus-based nano-systems on surfaces, (ii) probe selectively the catalytic and/or redox function of these nano-systems individually. By improving the resolution limit of this AFM-SECM technique, from the present 10 nm limit down to the ~1 nm range, it is expected that even individual enzyme molecules could be located on virus-scaffolds and interrogated one by one. This leap in resolution will rely on the know-how of Partner 3, a specialist of carbon nanotube manipulation and physics, who will be in charge of mounting sharpened multi–wall (and ultimately single-wall) nanotubes at the extremity of AFM-SECM probes.
The present project is truly interdisciplinary and puts in synergy concepts pertaining to modern enzymology, protein engineering, design and manipulation of functional nano-objects and spatially resolved biomolecular nanoelectrochemistry. The knowledge gathered will open up potential applications in nanocatalysis, lab on a chip and biosensor devices, drug delivery vectors and nanometrology.
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