Growing protein crystals is a tricky business, but their potential for use in drug development makes it worthwhile for researchers. The researchers study protein crystals using macromolecular crystallography, a powerful tool that reveals the structures and interactions of protein molecules. This information helps biomedical groups develop effective pharmaceutical compounds with shorter lead times and significantly lower development costs than those incurred using conventional drug development approaches (see sidebar).
To reduce some of the problems associated with protein crystal growth, groups like the one at the NASA-sponsored Center for Biophysical Sciences and Engineering at the University of Alabama (UAB; Birmingham, AL) are conducting their experiments in an unorthodox environment: space. The protein crystals used in the studies grow better in a microgravity environment that minimizes the effects of convection and sedimentation.1 Moving far beyond the boundaries of the familiar laboratory, the researchers now run the equivalent of a small aerospace company for designing and commissioning experiments as part of NASA's space shuttle and the international space station payloads. They have already sent crystal growth experiments up on more than 38 shuttle missions.
The UAB group has another experiment scheduled to launch this summer: a 115-day crystal growing experiment that will be performed aboard the international space station. The experiment will be controlled almost entirely from the ground.detecting clumps
The crystal growth process begins with supersaturated aqueous solutions. Under the proper conditions, the proteins in solution aggregate, then nucleate, and finally grow into crystals. Crystal growers want to control supersaturation, which affects growth, by regulating the solution concentration and temperature. Light from a diffused LED illuminates the area from the back. As crystallization occurs, a video camera records a stream of high-resolution images of the growing crystals. The research requires a noninvasive sensing system that can provide feedback to the control system that alters the rate of removal of water vapor, which in turn adjusts the crystal growth rate so that it provides fewer, larger crystals.
Conventional monitoring systems use video cameras to detect crystallization. Before crystallization takes place, however, the protein must aggregate. Ideally, crystal growers want to include this aggregation step in their feedback loop. To do this, the researchers use a laser scattering subsystem (LSS) to detect aggregation events earlier than is possible using conventional video analysis.
The scatterometry technique provides more precise control and optimization of the critical crystal growth process. In the UAB system, a collimated, fiber-delivered diode laser beam, shielded by a pinhole aperture, shines through the sample cell. The onset of aggregation, which heralds impending growth, increases the light scattered from the beam (see figure 1).2 A photodetector at 90° from the laser beam catches scattered light. Once aggregation begins, the crystal growth is monitored by the camera.
Figure 1. Protein aggregation triggers a slope change in the plot of detector voltage as a function of time. The computer system controlling the evaporation profile responds to this data to modify the growth, resulting in larger crystals.
For space-based operation, the LSS requires a stable diode laser light source and light delivery system (Point Source; Hamble, UK).2 The design presented three major challenges: the need for stable power output, athermal performance, and spatial stability of the beam. The challenge was to meet these requirements and maintain performance through the system without making design tradeoffs that would compromise performance. The rigors of space flight also require that the laser and delivery system be reliable because the cost implications of a malfunction are compounded by the fact that the equipment can't be repaired or replaced during a mission.
To fulfill the requirements, we built a pigtailed diode laser that delivers 2 mW at 670 nm via a miniature single-mode fiber delivery system. Overall, the laser provides output power stability of ±2% over a 12-hour period at an ambient temperature of 18°C.
Beam positioning is particularly important to the LSS operation. If the beam moves relative to the detector pinhole, the computer may mistake the change in the background scatter signal for the onset of aggregation. We designed collimators that were athermal, and a lens system that minimizes beam divergence using a 6-mm-diameter, 22-mm-long collimator to produce a 0.7-mm beam. The laser system's athermalized optomechanical design provides the research group with an output beam pointing error of less than 1 µrad/°C, and the output optics deliver a low divergence and low M2. A rugged Kevlar-lined, small-bore (3-mm outside diameter) stainless-steel jacket protects the polarization-maintaining fiber (see figure 2). The input optic is prefocused and optimized to achieve coupling efficiencies in excess of 70%.
Figure 2. A flexible but sturdy beam delivery system encases the polarization-maintaining fiber in a rugged Kevlar-lined small-bore stainless-steel jacket.
One of the most challenging aspects of the design was meeting footprint and weight restrictions. Payload is a cost factor for commercial organizations buying space and time on shuttle missions, so we had to build a system with the smallest possible footprint and at the lowest possible overall weight.
Because we had to make the entire system from materials approved by NASA with no outgassing, the system contains no plastics or outgassing adhesives. The laser system also has to withstand the extreme g-forces and vibrations experienced during launch and re-entry. We housed the equipment in a thermally controlled environment; the 800-mm fiber-optic cable allows remote location of the lasers to avoid any possibility of thermal disturbance to the crystallization process.
Photonics offers powerful solutions for space-based science. The technology provides compact, robust precision systems for monitoring delicate reactions. As certain types of manufacturing move into space, such systems may become commonplace in the future. oe
2. Terry L. Bray et al., J. Appl. Cryst. 31 (1998), pp. 515-522.
Probing protein crystals
Molecular biology seeks to understand the molecular basis of life by relating the structure of specific molecules such as proteins to their functional role in cells and organisms. In effect, the science was born with the discovery of the structure of DNA in 1953.
Proteins are of special interest to biologists, not only because of their complex structure, but also because of their variety and versatility. In the human body, there are as many as 100,000 different kinds of proteins that serve a multitude of purposes.
Recent advances have established protein crystallography as the lead instrument in structure-based drug design. The NASA-sponsored Center for Biophysical Sciences and Engineering at the University of Alabama (UAB; Birmingham, AL) is at the forefront of the groups using the structures of biological macromolecules to design pharmaceutical compounds. Center workers are pioneering research into dynamically controlled protein crystal growth methods. Researchers use the technique to study protein crystal growth in both terrestrial and microgravity environments.
Crystallography deals with the formation and properties of crystals, including the study of their growth, shape, and geometric character. A crystal has a definite, orderly atomic structure and an outward form bounded by smooth plane surfaces that are symmetrically arranged. Protein crystals, which contain 20% to 80% water by volume, are not generally stable outside of solution, but they can grow when a solid is formed gradually from a fluid.
Protein crystals can be identified by the shape and angle of their molecular crystals, despite differences in size or superficial differences in form. To determine the crystal structure of a substance, researchers use x-ray diffraction techniques in which the regular arrangement of the crystal acts as a distinctive grating.
Ken Green is OEM Design Manager at Point Source, Mitchell Point, Hamble, Hampshire, UK.