Understanding enzymatic function by advanced EPR

Summary

Protein biochemistry is the machinery that keeps living organisms running. At present, the structure of the assembled machine, the starting material and the products are known, but much less is understood about how the machine is assembled and how proteins perform specific and efficient catalysis. I propose to investigate this by combining modern biochemical techniques with instrumental development on the forefront of electron paramagnetic resonance (EPR). Specific questions concern (i) protein folding, (ii) protein-protein and protein-membrane interactions, and (iii) protein dynamics required for enzymatic function. These issues are of high biochemical impact: (i) Protein folding is the
assembly of the three-dimensional functional structure from the extended chain of aminoacids. To study protein folding with spin labels as paramagnetic probes, we will monitor the distance between a pair of spin labels during folding. (ii) Protein-protein and protein-membrane interactions are essential for biochemical reactions and wrong interactions may result in aggregation, which may cause diseases.
Protein-protein interactions will be studied on spin-labelled peptides prone for aggregation. (iii) To understand biochemical catalysis, the mechanism of enzyme reactions needs to be known. We will investigate enzyme reactions by detecting or trapping paramagnetic intermediates, and measuring protein dynamics during the reaction. The advantages of EPR are the exclusive sensitivity to paramagnetic centres, the high resolution to identify intermediates, the potential to determine electronic structure, and the large range of time and distance scales obtainable. To take full advantage of this potential, a variety of novel EPR techniques and methods of reaction kinetics will be combined.

Details

Project number

700.54.801

Main applicant

Prof. dr. E.J.J. Groenen

Affiliated with

Universiteit Leiden, Faculteit der Wiskunde en Natuurwetenschappen, Moleculaire Natuurkunde

Team members

Dr. M.I. Huber

Duration

01/01/2006 to 13/01/2010