(526d) Time Resolved Serial Protein Crystallography in a Microfluidic Device

Authors: 
Perry, S. L., UMass Amherst
Srajer, V., University of Chicago
Pawate, A. S., University of Illinois at Urbana-Champaign
Schieferstein, J. M., University of Illinois at Urbana-Champaign
Guha, S., University of Illinois at Urbana-Champaign
Kenis, P. J. A., University of Illinois at Urbana-Champaign

Renewed interest in room temperature diffraction has been prompted by the desire to observe structural dynamics of proteins as they function.  Serial crystallography, an experimental strategy that aggregates small pieces of data from a large, uniform pool of crystals, has been demonstrated at synchrotrons and X-ray free electron lasers.  We utilize a microfluidic crystallization platform for serial Laue diffraction from macroscopic crystals.  This strategy takes advantage of microfluidic integration and the exquisite control over transport phenomena possible at the microscale to enable the growth of a large number of high quality, isomorphous crystals for serial diffraction.  The ability to perform in situ diffraction directly in our microfluidic devices also eliminates the need for manipulation or harvesting of the fragile protein crystals, while serial data collection avoids challenges associated with radiation damage by enabling the collection of as little as a single frame of diffraction data from an individual crystal. 

Here we report the in situ time-resolved structural analysis of photoactive yellow protein (PYP), performed entirely in a microfluidic chip. Following laser-initiation, we observed the evolution of structural changes associated with the protein photocycle over timescales ranging from nanoseconds to milliseconds.  A complete dataset was obtained by merging small slices of Laue data taken from many individual crystals.  Electron density difference maps generated from merged data highlight the expected conformational changes of the chromophore with time, validating our approach. 

Looking forward, the ability of our microfluidic platforms to grow and collect in situ crystallographic data from a large number of isomorphous protein crystals has the potential to enable the use of synchrotron-based Laue diffraction for both serial crystallography, and the dynamic structural study of biochemical reactions that are functionally irreversible due to factors such as X-ray radiation damage, slow reversion back to the ground state, limitations of the crystal lattice, or the actual irreversible nature of the reaction.  Ultimately, the most significant potential of our microfluidics-based approach comes from the integration of fluid handling capabilities to enable flow-cell based triggering of enzymatic reactions for dynamic crystallographic analysis.