Terry L. Bray

Recent results and new hardware developments for protein crystal growth in microgravity

Abstract Protein crystal growth experiments have been performed on 16 space shuttle missions since April 1985. The initial experiments used vapor diffusion crystallization techniques similar to those used in laboratories for earth-based experiments. More recent experiments have assessed temperature-induced crystallization as an alternative method for growing high quality protein crystals in microgravity. Results from both vapor-diffusion and temperature-induced crystallization experiments indicate that protein crystals grown in microgravity may be larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

A fiber optic probe for monitoring protein aggregation, nucleation and crystallization

Protein crystals are often experimentally grown in hanging drops in microgravity experiments on-board the Space Shuttle orbiter. The technique of dynamic light scattering (DLS) can be used to monitor crystal growth processes in hanging droplets (∼ 30 μL) in microgravity experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we demonstrate that such experiments are now feasible. We apply a newly developed fiber optic probe to various earth and space (microgravity) protein crystallization system configurations to test its capabilities. These include conventional batch (cuvette or capillary) systems, a hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for temperature-induced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within a few seconds) means of quantitatively and non-invasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for performing single-angle static light scattering measurements. It utilizes extremely low levels of laser power (a few μW) and essentially eliminates the usual problems associated with optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for visualization of macroscopic protein crystals. This new optical diagnostic system makes possible the exploration of new ways to grow good quality crystals suitable for X-ray crystallographic analysis and may contribute to a concrete scientific basis for understanding the process of crystallization.

Real time evolution of concentration distribution around tetragonal lysozyme crystal: case study in gel and free solution

Two-beam Michaelson interferometry was used to study concentration gradient layers around gel-grown tetragonal lysozyme crystals. Crystals were grown in gel to depress convection and mimic microgravity. The evolution of the concentration profile near the growing surface, the width of the concentration layer, surface concentration, and concentration gradient were investigated and the correlation of these parameters with lysozyme crystal growth is discussed. The concentration gradient properties of gel-grown crystals were compared to those obtained for solution-grown crystals and were found to be different from their solution-grown counterparts. In particular, concentration gradients were wider and transport rate were slower for gel-grown crystals than for solution counterparts.

Crystal growth via computer controlled vapor diffusion

At present, the most popular protein crystallization method is vapor equilibration via the hanging drop method. This method, however, offers little control over the rate of equilibration between the growth solution and the reservoir. A device has been developed by the Center for Macromolecular Crystallography at the University of Alabama at Birmingham that offers precise control of vapor equilibration in the hanging drop method. We report herein qualitative crystallization data for selected proteins utilizing this device. These studies demonstrate that a slower controlled evaporation rate of protein growth solution generally resulted in smaller populations of larger protein crystals.

Protein crystal growth results from the United States Microgravity Laboratory-1 mission

Protein crystal growth experiments have been performed on fourteen space shuttle missions between April 1985 and June 1992. These space shuttle missions have been used to grow crystals of a variety of proteins using vapour diffusion, liquid diffusion, and temperature induced crystallization techniques. The United States Microgravity Laboratory-1 mission (june 25-July 9, 1992) was a space lab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in-orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. The hardware developed specifically for the USML-1 mission is discussed along with preliminary experimental results.

A preliminary study of space- and ground-grown insulin crystals by X-ray diffraction and by light scattering tomography

Abstract For the first time, both X-ray diffraction and light scattering tomography were used to study the same set of space- and ground-grown protein crystals. X-ray diffraction of the insulin crystals grown from T6 insulin solutions by temperature reduction showed that space-grown crystals diffract to higher resolution then those grown on the ground. The light scattering tomographs revealed that the space-grown crystals contained very few spherical micro-defects while the ground-grown ones had large numbers of extended micro-defects and dislocations. The tomographs also gave insight into the methods of defect formation. This study marks the beginning of qualification of light scattering tomograph as a tool for determining the suitability of a protein for use in X-ray diffraction studies.

Dynamic control of vapor diffusion protein crystal growth

Two laboratory based systems have been constructed to demonstrate methods which will allow for dynamic control of protein crystal growth. The technologies developed in these systems will be incorporated into future flight hardware for use in microgravity studies. The first system uses a precisely controlled vapor diffusion approach to monitor and control protein crystal growth. The system utilizes a humidity sensor and various interfaces under computer control to effect virtually any evaporation rate from up to 40 different growth solutions simultaneously. A second system utilizes the key features of the first system with the addition of a static laser light scattering sensor which can be used to detect aggregation events and trigger a change in the evaporation rate for a growth solution. This system also demonstrates that a Control/Follower configuration can be used to actively monitor one chamber and accurately control replicate chambers relative to the Control chamber. Results from experiments with bot...

Protein crystal growth studies at the Center for Macromolecular Crystallography

The Center for Macromolecular Crystallography (CMC) has been involved in fundamental studies of protein crystal growth (PCG) in microgravity and in our earth-based laboratories. A large group of co-investigators from academia and industry participated in these experiments by providing protein samples and by performing the x-ray crystallographic analysis. These studies have clearly demonstrated the usefulness of a microgravity environment for enhancing the quality and size of protein crystals. Review of the vapor diffusion (VDA) PCG results from nineteen space shuttle missions is given in this paper.

Protein Crystallography Services on the International Space Station

The Center for Macromolecular Crystallography (CMC) has performed protein crystal growth experiments on more than 30 U.S. Space Shuttle missions. Results from these experiments have clearly demonstrated that the microgravity environment is beneficial in that a number of proteins crystallized were larger or of higher quality than their Earth-grown counterparts. These microgravity results plus data from a variety of other investigators have stimulated various space agencies to support fundamental studies on macromolecular crystal growth processes. The CMC has devoted substantial effort toward the development of dynamically controlled crystal growth systems, which allow scientists to optimize crystallization parameters on Earth or in space. This capability plus the CMC’s experience in protein structure determination and structureguided drug development have attracted partnerships with a number of pharmaceutical and biotechnology companies. The CMC is currently designing a complete crystallographic laboratory for the International Space Station. This facility will support a variety of crystallization hardware systems, an X-ray diffraction rack for crystal characterization or complete X-ray data set collection, and a robotically controlled crystal mounting system with cryo-preservation capabilities. The X-ray diffraction rack and crystal harvesting/cryo-preservation systems can be operated with minimal crew time via telerobotic and/or robotic procedures. The CMC, in conjunction with its spin-off company, Diversified Scientific, Inc. (DSI), is currently marketing crystallography services, which include crystal growth, structure determinations and structure-guided drug development.