Keith Moffat
Research Summary / Selected Publications
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 10E13 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 %7E100 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.
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 10E13 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 %7E100 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...
Rajagopal, S., Anderson, S., Srajer, V., Schmidt, M., Pahl, R. and Moffat, K. A structural pathway for signaling in the E46Q mutant of photoactive yellow protein. Structure 13, 1-9 (2005)
Ihee, H., Rajagopal, S., Srajer, V., Pahl, R., Anderson, S., Schmidt, M., Schotte, F., Anfinrud, P.A., Wulff, M. and Moffat, K. Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. Proc.Nat.Acad.Sci.USA 102, 7145-7150 (2005)
Crosson, S., Rajagopal, S. and Moffat, K. The LOV domain family: Photoresponsive signaling modules coupled to diverse output domains. Biochemistry 42: 2-10 (2003)
Crosson S., and Moffat K. Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch. Plant Cell 14: 1067-1075 (2002)
Key, J. and Moffat, K. Crystal structures of deoxy and CO-bound bjFixLH reveal details of ligand recognition and signaling. Biochemistry 44, 4627-4635 (2005)
Schmidt, M., Rajagopal, S., Ren, Z. and Moffat, K. Application of singular value decomposition to the analysis of time-resolved macromolecular X-ray data. Biophys. J. 84, 2112-2129 (2003)
Srajer, V., Teng, T. Y., Ursby, T., Pradervand, C., Ren Z., Adachi, S., Schildkamp, W., Bourgeois, D., Wulff, M. and Moffat, K. Photolysis of the Carbon Monoxide Complex of Myoglobin: Nanosecond Time-Resolved Macromolecular Crystallography. Science 274: 1726-1729 (1996). For a review see Science 274, 1631-1632 (1996).
Baxter, R.H.G., Ponomarenko, N., Srajer, V., Pahl, R., Moffat, K. and Norris, J.R. Time-resolved crystallographic studies of light-induced structural changes in the photosynthetic reaction center. Proc.Nat.Acad.Sci.USA 101, 5982-5987 (2004)
Rajagopal, S., Anderson, S., Srajer, V., Schmidt, M., Pahl, R. and Moffat, K. A structural pathway for signaling in the E46Q mutant of photoactive yellow protein. Structure 13, 1-9 (2005)
Ihee, H., Rajagopal, S., Srajer, V., Pahl, R., Anderson, S., Schmidt, M., Schotte, F., Anfinrud, P.A., Wulff, M. and Moffat, K. Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. Proc.Nat.Acad.Sci.USA 102, 7145-7150 (2005)
Crosson, S., Rajagopal, S. and Moffat, K. The LOV domain family: Photoresponsive signaling modules coupled to diverse output domains. Biochemistry 42: 2-10 (2003)
Crosson S., and Moffat K. Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch. Plant Cell 14: 1067-1075 (2002)
Key, J. and Moffat, K. Crystal structures of deoxy and CO-bound bjFixLH reveal details of ligand recognition and signaling. Biochemistry 44, 4627-4635 (2005)
