The Faculty

Focus

Structural biology; time-resolved crystallography; molecular bases of signal transduction; photoreceptors; synchrotron radiation applications in biophysics; protein design

Education

B.Sc. Honors (Physics), University of Edinburgh, 1965; Ph.D. (Protein Crystallography), University of Cambridge, 1970

Research Summary

Our research concentrates in two areas: on the application of synchrotron radiation techniques to dynamic X-ray diffraction studies of macromolecules; and on the structures, mechanism and design of natural and engineered photoreceptors. With the advent of extremely intense, polychromatic, pulsed synchrotron X-ray sources, the X-ray exposure times required to record an excellent X-ray diffraction pattern have dropped dramatically to the microsecond time range and, in special cases, to 100 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? The short answer is "yes". However, ultrafast reaction initiation in the crystal is accomplished by a brief, intense laser pulse, which of course requires that the molecules be light-sensitive. We therefore began our studies on signaling photoreceptors of various classes. These reveal general principles e.g. that light sensor (input) and effector (output) functions are contained on different protein domains; and signaling photoreceptors appear to have evolved by domain fusion. Since not all interesting biological systems are light-sensitive we then asked: can we apply these general principles to confer sensitivity to light on othertwise light-insensitive systems, such as transcription factors or enzymes such as histidine kinases? Again the short answer is "yes". This leads to the development of powerful new tools for cell biology and biophysics, and extends the reach of optogenetics beyond natural photoreceptors such as channelrhodopsin to engineered photoreceptors, and beyond the neurosciences.

Selected Publications

Laue diffraction and time-resolved crystallography

  1. Moffat, K., Szebenyi, D.M.E. and Bilderback, D. (1984) X-ray Laue Diffraction from Protein Crystals.  Science 223, 1423-5.
  2. Srajer, V., Teng, T.Y., Ursby, T., Pradervand, C., Ren, Z., Adachi, S.I., Schildkamp, W., Bourgeois, D., Wulff, M. and Moffat, K. (1996) Photolysis of the Carbon Monoxide Complex of Myoglobin: Nanosecond Time-resolved Crystallography.  Science 274, 1726-9.
  3. Key, J., Srajer, V., Pahl, R. and Moffat, K. (2007) Time-resolved Crystallographic Studies of the Heme Domain of the Oxygen Sensor FixL: Structural Dynamics of Ligand Rebinding and Their Relation to Signal Transduction. Biochemistry 46, 4706-15.   
  4. Neutze, R. and Moffat, K. (2012)  Time-resolved Structural Studies at Synchrotrons and X-ray Free Electron Lasers: Opportunities and Challenges.  Curr. Opin. Struct. Biol. 22, 651-9.PMCID 3520507 
  5. Sugishima, M., Moffat, K. and Noguchi, M.  (2012) Discrimination Between CO and O2 in Heme Oxygenase: Comparison of Static Structures and Dynamic Conformation Changes Following CO Photolysis. Biochemistry 51, 8554-62. PMCID: PMC3548955
  6. Jung, Y.O., Lee, J.H., Kim, J., Schmidt, M., Moffat, K., Srajer, V., and Ihee, H. (2013) Volume-conserving Trans-Cis Isomerization Pathways in Photoactive Yellow Protein Visualized by Picosecond X-ray Crystallography.  2013 March; 5(3): 212–220. PMCID: PMC3579544

Signaling photoreceptors

  1. Crosson, S. and Moffat, K. (2001) Structure of a Flavin-binding Plant Photoreceptor Domain: Insights into Light-mediated Signal Transduction. Proc. Natl. Acad. Sci. USA 98, 2995-3000. 
  2. Yang, X., Stojkovic, E.A., Kuk, J. and Moffat, K.  (2007)  Crystal Structure of the Chromophore Binding Domain of an Unusual Bacteriophytochrome, RpBphP3, Reveals Residues that Modulate Photoconversion.  Proc. Natl. Acad. Sci. USA 104, 12571-12576. PMCID 1941510
  3. Toh, K.C., Stojkovic, E.A., van Stokkum, I.H.M., Moffat, K. and Kennis, J.T.M. (2010).  Proton Transfer and Hydrogen Bond Interactions Determine Fluorescence Quantum Yield and Photochemical Efficiency of Bacteriophytochrome  Proc. Natl. Acad. Sci. USA 107, 9170-5. PMCID 2889060
  4. Yang, X., Ren, Z., Kuk, J. and Moffat, K. (2011) Temperature-scanning Cryocrystallography Identifies Reaction Intermediates in Bacteriophytochrome. Nature 479, 428-32. PMCID 3337037

Design and engineering of photoreceptors

  1. Strickland, D., Moffat, K. and Sosnick T.  (2008)  Designed Allostery in a Modular Protein: Light-Activated DNA Binding.  Proc. Natl. Acad. Sci. USA 105, 10709-14. 
  2. Moeglich, A., Ayers, R. and Moffat, K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases.  J. Mol. Biol. 385, 1433-1444  
  3. Moeglich, A. and Moffat, K. (2010) Engineered Photoreceptors as Novel Optogenetic Tools.  Photochem. Photobiol. Sci. 9, 1286-1300.  
  4. Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K, and Moeglich, A. (2012)  From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.  J. Mol. Biol. 416, 534-42.
  5. Mitra, D., Yang, X., and Moffat K. (2012)  Crystal structures of Aureochrome1 LOV suggest new design strategies for optogenetics. Structure. 2012 Apr 4;20(4):698-706 PMCID: PMC3322389