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Structural enzymology: real-time tracking of enzyme catalysis in crystals

Objective

The use of single crystal UV/Vis and fluorescence spectroscopy has revolutionised the field of structural enzymology which takes advantage of the fact that many proteins retain their catalytic activity in the crystalline form and by soaking in substrates and substrate analogues it is possible to observe turnover in the crystal. In protein systems which undergo spectral changes in the UV or visible regions, single crystal UV/Vis spectroscopy can be used to track the formation of the intermediates thus generated. This information can be combined with fast-freeze trapping techniques to allow X-ray crystallographic structure determination of precisely identified reaction intermediates.

The current state-of-the-art is limited, however, to those systems with accessible chromophores. Recently, a single crystal Raman microscope has been described which allows, for the first time, real-time tracking of catalysis in crystalline proteins which do not possess UV or visible chromophores. However, the microscope setup is not ideal for the use of rapid freeze trapping techniques, as the crystal is suspended in a droplet of liquid over a sealed well. In this application, a new open-plan single crystal Raman spectrophotometer is proposed that is specifically designed to facilitate the use of rapid freeze trapping techniques for structural enzymology studies. The use of Raman spectroscopy opens the door to the application of cutting edge structural enzymology techniques to a greatly expanded range of biological systems, including those of medical and biotechnological importance.

Field of science

  • /natural sciences/chemical sciences/analytical chemistry/spectroscopy
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins

Call for proposal

FP6-2004-MOBILITY-12
See other projects for this call

Funding Scheme

IRG - Marie Curie actions-International re-integration grants

Coordinator

THE UNIVERSITY OF LEEDS
Address
Woodhouse Lane
Leeds
United Kingdom