Single-molecule fluorescent electrochemistry on redox proteins

Summary

Redox reactions in cells are of central importance in many metabolic pathways, for example respiration and photosynthesis. These reactions are controlled by redox proteins and enzymes.
In this research, it is proposed to develop a new approach for studying single redox proteins and enzymes by combining fluorescence and electrochemistry. This method will allow the study and manipulation of single electron transfer steps in redox proteins and of single-enzyme turnovers. This will provide new insights in the fundamentals of biological electron transfer and surface-biomolecule interaction.

The fluorescent detection in this method is based on redox-dependent FRET between a dye label and the redox center. This method is applicable to all redox proteins with a redox-state dependent absorption spectrum. Recently, I have achieved the first ?fluorescent electrochemistry?, the combination of fluorescent detection with potentiostatic control, for a monolayer of the redox protein azurin. By using potentiostatic control, detailed manipulation of the redox state is possible.

The observation of single electron transfer steps between protein and electrode, will show the time-dependence of redox properties for a single protein, the distribution of redox properties over several proteins, and heterogeneity in the interaction between protein and surface. None of these important issues can be investigated equally well with existing methods.

By applying the fluorescent electrochemistry to enzymes, a new, more general method will be added to the single-enzyme methods currently available. This will make it possible to show whether the conformational fluctuations observed in previous studies are a general feature of enzymes, and with the potentiostatic control, to correlate these fluctuations to the exact conditions of the enzymes.
As a bonus, extremely sensitive biosensors on the basis of such electronically accessible fluorescent proteins and enzymes are a possibility in the future.

Output

Scientific article

  • S Kuznetsova, N Akkilic, G Mizzon, T J Aartsma, J M Salverda, A V Patil, G W Canters, H A Heering, G Zauner, J J Davis(2010): Fluorescent voltammetry of immobilized azurin: a direct observation of thermodynamic and kinetic heterogeneity Angewandte Chemie International Edition pp. 5776 - 5779

Details

Project number

680-47-122

Main applicant

Dr. J.M. Salverda

Affiliated with

Universiteit Leiden, Faculteit der Wiskunde en Natuurwetenschappen, Leids Instituut voor Onderzoek in de Natuurkunde (LION)

Team members

Dr. J.M. Salverda

Duration

25/12/2007 to 23/02/2012