Structure-activity relationship of cytochromes-P450 studied by fluorescent labeling in the bulk and at the single-molecule level

The project for which we like to engage the participation of prof. S. Mazumdar, deals with the application of a technique developed in Leiden, to study the mode of action of cytochrome-P450..We have found that we can monitor enzyme activity by following the fluorescence of a dye label that has been covalently attached to the enzyme. The superior sensitivity and efficiency of optical detectors can therefore be used. It has been found that the principle is eminently suitable to obtain hitherto unavailable information about the functioning of complicated enzymes. Moreover, the technique carries the prospect of the implementation of enzymes into electronic devices like biosensors In the newest development the method has been shown also to be suitable for the study of single enzyme molecules. Cytochromes-P450 enzymes are wide-spread in nature and are involved in the conversion of a large variety of organic compounds that are potentially harmful to an organism. In humans they play a crucial role in detoxification and break-down of metabolites. Implementation of these enzymes into sensing devices (biosensors) could be of enormous importance for the development of new and fast high-throughput screening techniques to test potential drugs and pharmaceutical leads. Prof. Mazumdar is an internationally recognised authority on the enzymology of P450?s; his expertise will be indispenable for the efficient handling and labelling of the P450?s and the subsequent spectroscopic studies. He is willing to provide us with various forms of engineered human P450 enzymes and to assist in the labeling and the fluorescent studies of the labeled P450?s. The workplan consists of two phases: A. (4 weeks) Labeling of the P450?s with various dyes (Cy3, Cy5, ATTO dyes, etc.) and study their fluorescence behaviour in a fluoro-spectrophotometer under steady state conditions and by stopped flow techniques. B. (4 weeks) Study of the labeled enzyme after immobilization on a solid surface by means of confocal microscopy (single molecule conditions).