Titus J. Boggon

Protein Crystal Movements and Fluid Flows During Microgravity Growth

The growth of protein crystals suitable for X–ray crystal structure analysis is an important topic. The methods of protein crystal growth are under increasing study whereby different methods are being compared via diagnostic monitoring including charge coupled device (CCD) video and interferometry. The quality (perfection) of protein crystals is now being evaluated by mosaicity analysis (rocking curves) and X–ray topographic images as well as the diffraction resolution limit and overall data quality. Choice of a liquid—liquid linear crystal–growth geometry and microgravity can yield a spatial stability of growing crystals and fluid, as seen in protein crystallization experiments on the uncrewed platform EURECA. A similar geometry used within the Advanced Protein Crystallization Facility (APCF) onboard the crewed shuttle missions SpaceHab–01 and IML–2, however, has shown by CCD video some lysozyme crystal movement through the mother liquor. Moreover, spurts and lulls of growth of a stationary lysozyme protein crystal that was probably fixed to the crystal–growth reactor wall suggests g–jitter stimulated movement of fluid on IML–2, thus transporting new protein to the growing crystal faces. In yet another study, use of a hanging drop vapour diffusion geometry on the IML–2 shuttle mission showed, again via CCD video monitoring, growing apocrustacyanin C1 protein crystals executing near cyclic movement, reminiscent of Marangoni convection flow of fluid, the crystals serving as ‘markers’ of the fluid flow. These observations demonstrated that the use of vapour diffusion geometry did not yield spatially stable crystal position or fluid conditions for a solely protein diffusive regime to be realized. Indeed mosaicity evaluation of those vapour diffusion–grown apocrustacyanin C1 crystals showed inconsistent protein crystal quality, although the best crystal studied was microgravity grown. In general, realizing perfect conditions for protein crystal growth, of absence of movement of crystal or fluid, requires not only the correct choice of geometry but also the avoidance of low–frequency (≲5Hz) g–jitters. A review is given here of existing results and experience over several microgravity missions. Some comment is given on gel protein crystal growth in attempts to ‘mimic’ the benefits of microgravity on Earth. Finally, the recent new results from our experiments on the shuttle mission LMS are described. These results include CCD video as well as interferometry during the mission, followed, on return to Earth, by reciprocal space mapping at the NSLS, Brookhaven and full X–ray data collection on LMS and Earth control lysozyme crystals. Diffraction data recorded from LMS and ground control apocrustacyanin C1 crystals are also described.

Lysozyme crystal growth kinetics monitored using a Mach-Zehnder interferometer.

A Mach-Zehnder interferometer has been developed for the monitoring of the kinetics of the diffusion process in protein crystal growth. This device can be used in conjunction with the ESA Advanced Protein Crystallization Facility (APCF), which allows experiments under microgravity conditions (e.g. on board the NASA Space Shuttle). Experimental trials on the ground have been carried out with the interferometer using the engineering model of the APCF and a protein dialysis reactor. Chicken egg-white lysozyme crystal growth, as a test, has thereby been monitored directly. The changes of concentration in the solution over time have been determined via the refractive index measurements made and subsequently correlated with visual monitoring of crystal growth in a repeat experiment.

CCD video observation of microgravity crystallization of lysozyme and correlation with accelerometer data.

Lysozyme has been crystallized using the ESA Advanced Protein Crystallization Facility onboard the NASA Space Shuttle Orbiter during the IML-2 mission. CCD video monitoring was used to follow the crystallization process and evaluate the growth rate. During the mission some tetragonal crystals were observed moving over distances of up to 200 micrometers. This was correlated with microgravity disturbances caused by firings of vernier jets on the Orbiter. Growth-rate measurement of a stationary crystal (which had nucleated on the growth reactor wall) showed spurts and lulls correlated with an onboard activity: astronaut exercise. The stepped growth rates may be responsible for the residual mosaic block structure seen in crystal mosaicity and topography measurements.

Apocrustacyanin C1 crystals grown in space and on earth using vapour‐diffusion geometry: protein structure refinements and electron‐density map comparisons

Models of apocrustacyanin C(1) were refined against X-ray data recorded on Bending Magnet 14 at the ESRF to resolutions of 1.85 and 2 A from a space-grown and an earth-grown crystal, respectively, both using vapour-diffusion crystal-growth geometry. The space crystals were grown in the APCF on the NASA Space Shuttle. The microgravity crystal growth showed a cyclic nature attributed to Marangoni convection, thus reducing the benefits of the microgravity environment, as reported previously [Chayen et al. (1996), Q. Rev. Biophys. 29, 227-278]. A subsequent mosaicity evaluation, also reported previously, showed only a partial improvement in the space-grown crystals over the earth-grown crystals [Snell et al. (1997), Acta Cryst. D53, 231-239], contrary to the case for lysozyme crystals grown in space with liquid-liquid diffusion, i.e. without any major motion during growth [Snell et al. (1995), Acta Cryst. D52, 1099-1102]. In this paper, apocrustacyanin C(1) electron-density maps from the two refined models are now compared. It is concluded that the electron-density maps of the protein and the bound waters are found to be better overall for the structures of apocrustacyanin C(1) studied from the space-grown crystal compared with those from the earth-grown crystal, even though both crystals were grown using vapour-diffusion crystal-growth geometry. The improved residues are on the surface of the protein, with two involved in or nearby crystal lattice-forming interactions, thus linking an improved crystal-growth mechanism to the molecular level. The structural comparison procedures developed should themselves be valuable for evaluating crystal-growth procedures in the future.

Purification, crystallization and initial X-ray analysis of the C1 subunit of the astaxanthin protein, V600, of the chondrophore Velella velella

The subunit C1 of the carotenoid-binding protein, V600, of the chondrophore Velella velella has been purified and crystallized. The crystals, which were grown by the vapour-diffusion method from ammonium sulfate as the major precipitant, diffract beyond 3 A and show little radiation damage over long periods (greater than 100 h) on a Cu Kalpha rotating-anode X-ray source. The space group of the crystals is P212121 with cell dimensions a = 42.0, b = 80.9, c = 110. 6 A.