Research

Microfluidic Platforms for Time-Resolved Crystallography

Efforts to understand structure-function relationships in proteins and other biomolecules have relied heavily on static structural depictions obtained via traditional monochromatic X-ray crystallography. However, such data are unable to fully describe the dynamic nature of enzymatic processes. Reasons for this limitation include; (i) the fact that the timescale for data collection using monochromatic X-rays (seconds) is orders of magnitude longer than the timescale over which structural changes and short-lived reaction intermediates occur (femtoseconds to milliseconds), (ii) protein crystallography is typically performed at cryogenic temperatures which limit the extent of molecular motion, and (iii) the need to repeatedly trigger the enzymatic reaction within the crystal to obtain a complete dataset.

While traditional monochromatic X-ray diffraction is used for the vast majority of structural biology studies because of the relative simplicity of data analysis, polychromatic, or Laue diffraction, enables significantly faster data collection by dramatically increasing the flux of X-rays at the sample and increasing the density of data obtained per frame. At the BioCARS Laue beamline at the Advanced Photon Source at Argonne National Laboratory it is possible to collect diffraction data from the X-rays emitted by a single bunch of electrons travelling around the synchrotron ring. This enables the collection of data at a time resolution of ~100 ps, compared to a time-scale of seconds for monochromatic experiments.

Unfortunately, the speed benefits attained with Laue diffraction are counterbalanced by significant challenges associated with radiation damage. In order to obtain a complete structural depiction of a protein it is necessary to collect tens, if not hundreds of frames of data, depending upon the desired number of time points. It is also necessary to perform this data collection at biologically-relevant temperatures, rather than at cryogenic conditions where the motion of the enzyme and the effects of radiation damage can be frozen in place. These harsh conditions have meant that time-resolved Laue analysis has only been a viable strategy for proteins where extremely large, stable crystals can be grown such that data could be collected from multiple locations on the crystal. Recently, however, the idea of serial crystallography, where only a single frame of data is collected per crystal, has been popularized with the introduction of X-ray free electron lasers (XFELs) as incredibly brilliant sources of X-rays. The efficient extension of serial crystallography to Laue methods has tremendous potential to enable the time-resolved structural analysis of classes of proteins that had previously been considered inaccessible to these methods, including receptors, drug targets, and metabolic enzymes.

Highly integrated microfluidic networks can easily be designed to grow a large number of protein crystals for use in serial crystallography (see Figure). Integration, coupled with small length-scales enables the parallel formulation of samples while using miniscule sample volumes in an environment free of inertial and convective effects. This exquisite control over local concentrations and gradients has been shown to facilitate reproducible sample formulation, including the growth of large numbers of isomorphous crystals, and can also facilitate reproducible chemical triggering of enzymatic reactions (i.e., ligand addition) for analysis; challenges that are difficult to address using traditional, methods. Such chips can also enable high throughput dynamic structural analysis with respect to multiple variables including pH, ionic strength, and ligand concentration for more complex analyses.

Funding

Collaborations

References

Patents