Fluorescence energy transfer enzyme conformation, changes

The application of total internal reflection fluorescence spectroscopy (TIRF) by this laboratory to the study of protein adsorption at solid-liquid interfaces is reviewed. TIRF has been used to determine adsorption isotherms and adsorption rates from single-and multi-component protein solutions. Initial adsorption rates of BSA can be explained qualitatively by the properties of the adsorbing surface. Most recently, a TIRF study using monoclonal antibodies to probe the conformation of adsorbed sperm whale myoglobin (Mb) elucidated two aspects of the Mb adsorption process 1) Mb adsorbs in a non-random manner. 2) Conformational changes of adsorbed Mb, if they occur, are minor and confined to local regions of the molecule. Fluorescence energy transfer and proteolytic enzyme techniques, when coupled with TIRF, can characterize, respectively, the conformation and orientation of adsorbed Mb. 

The metal-NADH distance determined by fluorescence studies on the cobalt-substituted enzyme, using binary NADH, thio-NADH, and Rose Bengal/enzyme complexes, differs from the above.242 The minimum metal-NADH distance determined by Forster calculations based on the energy transfer from bound ligand to cobalt(n) is reported as 19 A. It is thought possible that the discrepancy between this value and that determined by X-ray diffraction may be due to conformational changes in solution. 

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