Structural and structure-function research on complex biomolecular machinery is mostly restricted to studies of states rather than processes due to the fragile and rapid nature of interactions between participating molecules. Methods currently used for kinetic studies provide either good time resolution or good distance resolution but not both. Time-resolved photoaffinity crosslinking can provide direct angstrom-scale measurement of conformational changes within functioning macromolecular complexes. This method is a powerful tool for studies of rapid inter- and intramolecular movements in solution. The data are independent of the enzymatic reaction product(s) and the method can be applied to study essentially any multi-component biomolecular system. Reverse transcriptase is an important target for the development of new antiretrovirals (e.g. anti-AIDS drugs). Conformational changes of HIV-RT during polymerization occur on a sub-millisecond time scale and can only be studied by means of the rapid time-resolved techniques. We propose to create a robotic (fully automated) instrument for the time-resolved photoaffinity crosslinking and a set of heterobifunctional photoactivatable carbene-generating reagents, and test this system in our studies of conformational changes in retroviral HIV-1 reverse transcriptase (RT) during its enzymatic activity. The system will be broadly applicable to functional topography studies of a variety of macromolecular complexes that are crucial for our understanding of cancer and other diseases. ? ?
