Our research concentrates on the application of synchrotron radiation techniques to dynamic X-ray diffraction studies of macromolecules.

For the past 60 years, crystallographers have used monochromatic X-rays almost exclusively, that yield a static, time- and space-average structure of crystals of both small and large molecules.  The time-average is taken over the duration of the X-ray exposure, which is typically several hours with laboratory sources; and the space-average over all the molecules in the crystal, typically 1013 in number.  With the advent of extremely intense, polychromatic, pulsed synchrotron X-ray sources, the X-ray exposure times have dropped dramatically to the millisecond time range and, in special cases, to 150 picoseconds.  That is, exposure times are now comparable with the lifetime of biochemical intermediates in such fundamental processes as enzyme catalysis, ligand-binding and release, and photocycling in light-sensitive systems.  The question then arises: Can we generate such intermediates in the crystal and follow the evolution of their tertiary structures as the reaction proceeds, through observation of changes in the X-ray diffraction intensities?  Attacking the question requires suitable X-ray optics, cameras, detectors, computer software, and crystallographic theory, which can then be applied to studying crystals that respond to photoactivation, to a temperature or pressure jump, or to diffusion into the crystal of a substrate or reactant.

Over the past 15 years, we have developed the theoretical foundation of polychromatic (Laue) x-ray diffraction from single crystals, and the experimental protocols, hardware and software necessary to apply this diffraction technique to nanosecond time-resolved crystallography.  We have successfully introduced the time domain into crystallography, hitherto regarded as purely a static technique.  We initially applied this time-resolved, ultra-fast structural approach to studies of heme and globin relaxation and ligand rebinding in heme proteins such as myoglobin, and to the light-driven, structural response in photoactive proteins.  With successful results in hand, we are now extending this approach to several other light-sensitive signaling systems that are chemically and biologically diverse; and developing new techniques that will enhance the time resolution from the nanosecond range, first to a few hundred picoseconds and perhaps ultimately to femtoseconds.

Through the Consortium for Advanced Radiation Sources (CARS), we have developed synchrotron X-ray beam lines suitable for these and many other experiments in structural biology at the Advanced Photon Source (APS), at Argonne National Laboratory.

Keith Moffat