Deriving detailed structure and dynamics information for macromolecules and their complexes is a challenging and important step towards providing a molecular understanding of normal and malignant cells. New techniques of spectroscopy, microscopy, and crystallography have been developed over the last 10 years to address these questions. However, understanding the structure and dynamics of macromolecular assemblies is a problem of increasing importance. Although existing biophysical methods, such as fluorescence, single molecule techniques, crystallography and NMR have advanced our understanding greatly, it has been difficult to achieve high structural resolution, fast time-resolution, and the ability to monitor large assemblies while utilizing small amounts of precious samples. Time-resolved synchrotron x-ray footprinting is a relatively new technique developed to study the dynamics of nucleic acids. The method probes the solvent accessible surface of macromolecules and their complexes using hydroxyl radicals. The technique is coupled to stopped-flow initiation of reactions, and dynamics on timescales as fast as 5 milliseconds have been probed for nucleic acids. The technique has not yet been applied to analyzing the dynamics of proteins and their assemblies, or their complexes with nucleic acids. In the R21 phase of this proposal, we will develop a quantitative hydroxyl radical """"""""footprinting"""""""" technique using synchrotron radiation to probe the structure of proteins. Successful examination of protein folding and protein-ligand complexes will provide milestones for the transition to the second phase of the grant. In the R33 phase, we will further develop the footprinting techniques to allow detailed examination of interactions of large macro molecular complexes. Specifically, the focus will be methodologies to examine the detailed pair-wise interactions of large binary complexes from the individual perspective of each member of the pair. The model systems used to develop the technology include: 1) examining the orientation and binding of cofilin and actin, 2) examining the time-resolved dynamics of actin filament disassembly catalyzed by gelsolin, and 3) probing the time-resolved dynamics of reverse transcriptase. These model systems will """"""""drive"""""""" the technology to provide general methods relevant to studying a wide range of problems in cancer biology. Time-resolved protein footprinting, when perfected, will be applicable to studying macromolecular interactions critical to replication, transcription, signal transduction, translation, processing, and secretion.
