Acta Cryst. (1996). D52, 529-533 529 Lysozyme Crystal Growth Kinetics Monitored Using a Mach-Zehnder Interferometer E. H. SNELL,at J. R. HELLIWELL, a T. J. BOGGON, a P. LAUTENSCHLAGER b AND L. POTTHASTb aChemistry Department, University of Manchester, Oxford Road, Manchester M13 9PL, and bDornier GmbH, Raumfahrt-Infrastruktur, 88039 Friedrichshafen, Germany. E-mail: hell@man.ac.uk (Received 14 June 1995; accepted 8 January 1996) Abstract 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 experi- ments 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 corre- lated with visual monitoring of crystal growth in a repeat experiment. I. Introduction For the detailed understanding of protein crystal growth as a process it is necessary to perform diagnostic and monitoring experiments. Following the nucleation stage it is possible to consider monitor- ing, as a function of time, the changes in concentration in a solution of a precipitant agent and a protein by use of interferometry. Hence, a quantitative representation of the process of diffusion of protein and salt in a crystal growth chamber can be obtained. Such information should be useful to quantify the effect of differing chemical agents as well as physical parameters (e.g. temperature, gravity) on the crystal growth process in terms of fluid flow. Different types of interferometer can be consid- ered for the study of the refractive index changes to be expected in a protein crystal growth solution (Shlichta, 1986). These are notably the Michelson interferometer and the Mach-Zehnder interferometer designs. The advantage of the latter type is its compactness making it particularly suitable for t Current address: NASA Laboratory for Structural Biology, Code ES76, Building 4464, Marshall Space Flight Center, Huntsville, AL 35812, USA. ,~_~ 1996 International Union of Crystallography Printed in Great Britain - all rights reserved incorporation into existing apparatus used for micro- gravity experimentation e.g. the ESA Advanced Protein Crystallization Facility (APCF) developed for and now flown frequently on board NASA's Space Shuttle. The APCF (Snyder, Fuhrmann & Walter, 1991; Bosch, Lautenschlager, Potthast & Stapelmann, 1992) was constructed to provide a wider range of crystallization methods under microgravity than hitherto. It provides free-interface diffusion, dialysis and vapour-diffusion protein crystal growth reactors. The APCF flew, for example, on the STS-57 Spacehab-1 and STS-65 IML-2 Shuttle missions. To assess protein crystal perfection under microgravity growth, X-ray diffraction measurements were made on lysozyme crystals grown in dialysis reactors on the STS-57 and STS-65 missions (Helliwell, Snell & Weisgerber, 1995). The results showed improvements of a factor of 3-4 in mosaicity for each mission over the associated ground controls (Snell et al., 1995). Differences in crystal perfection between the two missions were observed however and raise the question of what is the actual optimum length of time, under microgravity conditions, for crystal growth to yield the best crystal quality. Moreover, even in the shorter mission (5 d), which yielded the best degree of perfection, there was still some imperfection manifest when comparing the measured mosaicity versus the theoretical limit (Snell et al., 1995; Helliwell, 1988). Hence, interferometry diag- nostic monitoring through a microgravity mission offers the prospect of restricting crystal growth to a period shorter than a mission duration i.e. so as to optimize the perfection of crystals. In the develop- ment of a Mach-Zehnder interferometer for the APCF it is essential to first undertake trials with it on the ground. Lysozyme was chosen as a test sample because our perfection measurements and calculations have been made with this system. Also, much is already known about it and it can be easily crystallized. This paper describes ground crystal- lization trials and studies with lysozyme using the engineering model of the APCF fitted with the Mach-Zehnder interferometer and the results it has produced. Acta Crystallographica Section D ISSN 0907-4449 ©1996