Structural Biology

Crystallization of difficult to purify proteins and large complexes. The first commercial product resulting from our microfluidic technology is a chip that allows optimal protein crystal growth with tiny amounts of sample; this device recently won an industry award for best new proteomics product. It was developed in collaboration with the structural biologist James Berger (U.C. Berkeley) (1). There are many other ways to exploit the unique physics of the microfluidic environment for protein crystal growth and analysis; this will continue to be a significant component of my future research program. For example, we have recently shown that it is possible to use microfluidic devices to perform extensive surveys of the phase behavior of proteins, and that this knowledge can be used to create “rational” crystallization screens that outperform conventional screens by a wide margin (2). Figure 2 shows a possible workflow for microfluidic structural biology using these tools. We believe it will be possible to develop microfluidic devices that perform in vitro directed mutations on cloned genes, followed by in vitro or cell-based protein expression, purification and solubility screening in a completely integrated fashion. We also believe that microfluidic devices will facilitate solving structures of large, multi-component complexes by allowing well controlled, systematic and exhaustive exploration of the various possible combinatoric crystallization experiments.

Figure 2: Diagram illustrating how various microfluidic devices developed in the Quake group can be used in various stages of the protein structure determination process. Although there are many possible scenarios, we describe one path in detail. Broad Search: the microfluidic formulator is used to screen thousands of chemical conditions and to determine the phase behavior of the most promising. 10 From this information, a rational or “smart” screen is developed. The crystallization screen is performed using the Free Interface Diffusion chip, which takes advantage of special physics of the microfluidic environment to achieve nearly optimal crystallization kinetics. 6 Once a hit is discovered and optimized, the conditions are reproduced in a growth chip. A segment of this chip can be punched out so that the cryoprotectant is added and x-ray diffraction patterns are taken in situ , without direct handling of the crystal. (unpublished).

1. C.L. Hansen, E. Skordalakes, J.M. Berger, and S.R. Quake, "A Robust and Scalable Microfluidic Metering Method that Allows Protein Crystal Growth by Free Interface Diffusion", Proc. Nat'l Acad. Sci. 99: 16531-6 (2002).

2. C.L. Hansen, M.O.A. Sommer, and S.R. Quake, Proc. Nat'l Acad. Sci., in press.