Time-resolved X-Ray Scattering of Proteins in Solution: the Pros and Cons of an ERL
Philip Anfinrud
Laboratory of Chemical Physics, NIH/NIDDK
The use of synchrotron radiation to solve protein structures has been enormously successful. However, the high-salt, crystalline environment in which structures are determined is quite unnatural. For example, crystal packing forces can restrain the conformational flexibility of the protein. Indeed, it is this flexibility that enables proteins to perform their designed tasks with such exquisite efficiency and selectivity. To deliver oxygen efficiently from the lungs to the tissues, tetrameric hemoglobin undergoes a cooperative R-state (high-affinity) to T-state (low-affinity) quaternary structure transition while passing through oxygen-poor capillaries. This structural transition is not accommodated by the crystal, and when executed, the crystal cracks. Consequently, structural studies of hemoglobin have thus far have focused only on the end points of the quaternary structure transition. The next frontier in structural biology is not amassing more static structures of proteins, but will be in developing an understanding of how they function in mechanistic detail, i.e., the time-ordered sequence of structural events that connect the initial and final states. Clearly, such studies require time-resolved methods. We recently developed the technique of 150-ps time-resolved Laue crystallography and used this method to study structural changes in proteins on the picosecond to millisecond time scale. To study processes that cannot occur in a crystal, we are currently developing the method of time-resolved X-ray scattering. We take advantage of the fact that the radial intensity distribution of the scattered X-ray photons is related to the size, shape, and structure of the protein. Because this technique provides one-dimensional data, assigning time-resolved scattering patterns to specific structural transitions requires much help from theory. One might suspect that this method will be sensitive only to large amplitude changes; however, our preliminary time-resolved studies have shown that this method is sensitive to the subtle tertiary conformational changes that occur when photolyzed carbon monoxy myoglobin transitions from its carboxy to its deoxy state. Most of these changes are too fast to be resolved with the 150-ps time resolution available on current 3rd generation synchrotrons. The parameters proposed for an ERL would allow this technique to probe this structural transition on the time scale in which it occurs, and would even access the chemical time scale of femtoseconds. The parameters required to pursue time-resolved X-ray scattering measurements on an ERL will be discussed.
