(508d) Microfluidic Platforms for Time Resolved Laue Crystallography

Microfluidic chips for combinatorial analyses such as protein crystallization have advanced tremendously in the past decade.  These platforms take advantage of parallel processing and small volumes, while creating an environment free of inertial or convective effects and providing exquisite control over local conditions and gradients. The ability to create highly reproducible microenvironments on chip has proven to be a tremendous asset for improving the consistency and quality of protein crystals grown on-chip. Coupling these benefits with advanced in situ protein crystallography techniques, microfluidic technology is now poised to facilitate a wide range of structure-function studies than have previously been accessible.

Standard X-ray diffraction methods are performed at cryogenic conditions that freeze out enzyme motion and collect frames of data on a time-scale of seconds.  However, polychromatic Laue crystallography enables ultra-fast data collection at biologically relevant temperatures, matching the timescale of analysis to the chemical and structural changes in the protein.  Two challenges associated with time-resolved Laue data collection are (i) the severe radiation damage associated with room temperature data collection using an intense polychromatic X-ray beam and (ii) the need to simultaneously trigger all of the protein molecules in a crystal for diffraction analysis. 

We use high throughput X-ray transparent microfluidic platforms for protein crystallization to address these challenges.  The fine control over composition on-chip facilitates the easy growth of a large number of isomorphous crystals. X-ray diffraction analysis of these crystals can then be performed in a serial fashion, collecting only a small amount of data from each individual crystal so as to avoid radiation damage. Here we report the in situ time-resolved structural analysis of photoactive yellow protein from protein crystals grown and analyzed on-chip. The reaction is laser-initiated and diffraction images are collected at time delays ranging from nanoseconds to milliseconds. Electron density difference maps generated from merged data can be used to clearly track the expected conformational changes of the chromophore with time, validating our approach. Future efforts will be focused on extending these capabilities to enable chemical triggering of enzymatic reactions (i.e. ligand addition) through the development of crystallization arrays containing flow-cell geometries.