Scientific approach to the optimization of protein crystallization conditions for microgravity experiments

The National Space Development Agency of Japan (NASDA) developed a practical protocol to optimize protein crystallization conditions for microgravity experiments. This protocol focuses on the vapor diffusion method using high density protein crystal growth (HDPCG)--hardware developed by the University of Alabama, Birmingham--that flew on the STS-107 mission. The objective of this development was to increase the success rate of microgravity experiments by setting crystallization conditions based on knowledge of crystal growth and fluid dynamics. The protocol consists of four steps: (1) phase diagram preparation, (2) estimation of condensation rate in the vapor diffusion method, (3) fluid dynamic property measurement, and (4) fluid dynamic simulation. First, a phase diagram was constructed. Crystallization characteristics were investigated by a microbatch method. The data were recalculated based on classical nucleation theory and the crystallization boundary was determined as a function of time. The second step was to develop a practical model to estimate the condensation rate of the crystallizing solution, including protein and precipitant, as a function of the precipitant concentration and solution volume. By considering the crystallization map and the vapor diffusion condensation model we were able to optimize the crystallization conditions that generate crystals in the desired time. This was particularly important in a shuttle mission whose mission duration is limited. The third step was fluid dynamic property measurement necessary for fluid dynamics simulation and crystal growth study. The last step was to estimate the mass transport in space on the basis of the fluid dynamics simulation transport model. It turned out that neither the vapor phase nor the solution phase was seriously affected by gravity until nucleation provided the hardware was set in a normal direction. Therefore, we concluded that the optimized crystallization conditions could be directly applied to microgravity experiments. By completing the approach, we were able to control the time for nucleation in the vapor diffusion method.