Search

We show that knowledge of the phase behavior of a protein allows one to create a rational screen that increases the success rate of crystallizing challenging proteins. The strategy is based on using microfluidics to perform large numbers of protein solubility experiments across many different chemical conditions to identify reagents for crystallization experiments. Phase diagrams were generated for the identified reagents and used to design customized crystallization screens for every protein. This strategy was applied with a 75% success rate to the crystallization of 12 diverse proteins, most of which failed to crystallize when using traditional techniques. The overall diffraction success rate was 33%, about double what was achieved with conventional automation in large-scale protein structure consortia. The higher diffraction success rates are achieved by designing customized crystallization screens using the phase behavior information for each target. The identification of reagents based on an understanding of protein solubility and the use of phase diagrams in the design of individualized crystallization screens therefore promotes high crystallization rates and the production of diffraction-quality crystals.

Microfluidic technologies promise unprecedented savings in cost and time through the integration of complex chemical and biological assays on a microfabricated chip. Recent advances are making elements of this vision a reality, facilitating the first large-scale integration of microfluidic plumbing with biological assays. The power of miniaturization lies not only in achieving an economy of scale, but also in exploiting the unusual physics of fluid flow and mass transport on small length scales to realize precise and efficient assays that are not accessible with macroscopic tools. Diverse applications ranging from time-resolved studies of protein folding to highly efficient protein crystal growth suggest that microfluidics may become an indispensable tool in biology.

The article reports on the manufacture of a microfluidic device for kinetic optimization of protein crystallization and structure determination in the U.S. The device provides a link between chip-based nanoliter volume crystallization screening and structure analysis. In addition, the device allows control over the rate and extent of water vapor transport through a permeable membrane.