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Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Design of Light-sensitive Molecules for Time-resolved Crystallography and Optogenetics

Time-resolved crystallography uses the brief, intense X-ray pulses emitted by synchrotron or hard X-ray free electron laser (FEL) sources to probe the time course of structural changes as they occur in the molecules in a crystal, via pump - probe experiments. Synchrotron sources such as BioCARS sector 14 at the Advanced Photon Source, Argonne National Laboratory, can access the time scale from seconds to 100 picoseconds, where the lower limit is set by the duration of a single X-ray pulse. The new FEL sources such as the Linac Coherent Light Source at Stanford extend this limit to femtoseconds. The characteristics of the X-ray pulses emitted by synchrotron and FEL sources are radically different (Neutze & Moffat, 2012), which necessitates new approaches to the pump - probe experiments and data analysis. For both synchrotron and FEL sources, reaction initiation typically requires light-sensitive systems in which reaction can be initiated by a visible laser pulse - but clearly, not all interesting biological systems are light-sensitive. This raises the question: how can sensitivity to light be conferred on otherwise light-inert systems, by optogenetic approaches?

Ultrafast time-resolved crystallography will be illustrated by experiments conducted at synchrotron (Jung, Lee, Kim, Schmidt, Moffat, Srajer & Ihee, 2013) and FEL (Tenboer et al., 2014) sources, that probe the structure of short-lived structural intermediates in the photocycle of the naturally-occurring bacterial blue light photoreceptor known as photoactive yellow protein, PYP. The principles that can be used to confer sensitivity to light on light-inert systems (Moeglich & Moffat, 2010; Moffat, 2014) are illustrated by the design and characterization of a blue-light-sensitive histidine kinase (Moeglich, Ayers & Moffat, 2009). Finally I ask: are these principles equally applicable to experiments in the femtosecond time range at FEL sources?

This research has been supported by NIH grants GM111072 and EY024363.

References
    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. Nature Chem. 5, 212-20.

    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.

    Moeglich, A., Ayers, R. and Moffat, K. (2009). Design and signaling mechanism of light-regulated histidine kinases. J. Mol. Biol. 385, 1433-1444

    Moeglich, A. and Moffat, K. (2010). Engineered photoreceptors as novel optogenetic tools. Photochem. Photobiol. Sci. 9, 1286-1300.

    Moffat, K. (2014). Time-resolved crystallography and protein design: signaling photoreceptors and optogenetics. Phil. Trans. R. Soc. B 369: 2013568.

    Tenboer, J., Basu, S.,, Zatsepin, N., Pande, K., Milithianaki, D., Frank, M., Hunter, M., Boutet, S., Williams, G.J., Koglin, J.E., Oberthuer, D., Heymann, M., Kupitz, C., Conrad, C., Coe, J., Roy-Chowdhury, S., Weierstall, U., James, D., Wang, D., Grant, T., Barty, A., Yefanov, O., Scales, J., Gati, C., Seuring, C., Srajer, V., Henning, R., Schwander, P., Fromme, R., Ourmazd, A., Moffat, K., Van Thor, J.J., Spence, J.H.C., Fromme, P., Chapman, H.N. and Schmidt, M. (2014). Time-resolved serial crystallography captures high resolution intermediates of photoactive yellow protein. Science 346, 1242-6.


Keith Moffat

Department of Biochemistry & Molecular Biology
Institute for Biophysical Dynamics and Center for Advanced Radiation Sources
The University of Chicago
Chicago, IL 60637

Ph: (773) 702-2116
Fax: (773) 702-0439
moffat@cars.uchicago.edu