(83c) Microfluidic Platforms for Time Resolved Laue Crystallography

While efforts in microfluidics for protein crystallization have developed dramatically in the past ten years [1-3], the idea of coupling microfluidic technology with advanced protein crystallography techniques for in situ analysis is an area where significant advances can be made.  Microfluidic devices are particularly applicable for Laue methods [4], where polychromatic X-rays allow for significantly faster data collection that enables the analysis of tiny or fragile crystals that are susceptible to radiation damage as well as kinetic experiments [5,6].  This high rate of data collection enables time-resolved crystallography experiments by matching the X-ray exposure time to the timescale of chemical and structural changes in the protein.  Thus Laue crystallography provides a very elegant platform for performing kinetic experiments that directly probe changes that occur during enzymatic function. 

Microfluidic platforms have the benefit of not only enabling experiments at very small volumes, but also by creating an environment free of inertial or convective effects and allowing for exquisite control over local conditions and gradients.  We have developed microfluidic platforms for protein crystallization which utilize this high degree of control for both the growth of high quality protein crystals, and also as a triggering mechanism for use in time resolved crystallography experiments.  Here we report on microfluidic crystallization platforms that have been optimized for the growth of a large number of crystals for in situ time resolved Laue analysis.   We used photoactive yellow protein (PYP) as a model system for the first time resolved crystallography experiments.  The enzymatic reaction is first triggered by a laser light pulse and then a series of X-ray diffraction images are taken at varying time delays.  This large set of diffraction data is then analyzed to yield a time resolved picture of the various structural changes within the crystal.  We are working on extending the capabilities of our microfluidic systems to include triggering of enzyme reactions by flowing a solution over the crystals.  This capability will enable the time resolved structural analysis of a large range of protein systems, which had previously been inaccessible for study. 

1.         Hansen, C. and S.R. Quake, Microfluidics in structural biology: smaller, faster... better. Current Opinion in Structural Biology, 2003. 13(5): p. 538-544.

2.         van der Woerd, M., D. Ferree, and M. Pusey, The promise of macromolecular crystallization in microfluidic chips. Journal of Structural Biology, 2003. 142(1): p. 180-187.

3.         Perry, S.L., et al., Microfluidic Generation of Lipidic Mesophases for Membrane Protein Crystallization. Crystal Growth & Design, 2009. 9(6): p. 2566-2569.

4.         Ren, Z., et al., Laue crystallography: coming of age. Journal of Synchrotron Radiation, 1999. 6: p. 891-917.

5.         Cornaby, S., et al., Feasibility of one-shot-per-crystal structure determination using Laue diffraction. Acta Crystallographica Section D, 2010. 66(1): p. 2-11.

6.         Bourgeois, D., et al., Time-resolved methods in biophysics. 6. Time-resolved Laue crystallography as a tool to investigate photo-activated protein dynamics. Photochemical & Photobiological Sciences, 2007. 6(10): p. 1047-1056.