Effect of macromolecular mass transport in microgravity protein crystallization

Abstract

To investigate the effect of macromolecular transport and the incorporation of protein aggregate impurities in growing crystals, experiments were performed on the International Space Station (ISS) and compared with control experiments performed in a 1G laboratory environment. Crystal growth experiments for hen egg-white lysozyme (HEWL) and Plasmodium falciparum glutathione S-transferase (PfGST) were monitored using the ISS Light Microscopy Module (LMM). Experiments were performed applying the liquid–liquid counter diffusion crystallization method using rectangular, optically transparent capillaries. To analyze the quantity of impurity incorporated into growing crystals, stable fluorescently labeled protein aggregates were prepared and subsequently added at different percent concentrations to nonlabeled monomeric protein suspensions. For HEWL, a covalent cross-linked HEWL dimer was fluorescently labeled, and for PfGST, a stable tetramer was prepared. Crystallization solutions containing different protein aggregate ratios were prepared. The frozen samples were launched on 19.02.2017 via SpaceX-10 mission and immediately transferred to a -80°C freezer on the ISS. Two series of crystallization experiments were performed on ISS, one during 26.02.2017 to 10.03.2017 and a second during 16.06.2017 to 23.06.2017. A comparison of crystal growth rate and size showed different calculated average growth rates as well as different dimensions for crystals growing in different positions along the capillary. The effect of macromolecular mass transport on crystal growth in microgravity was experimentally calculated. In parallel, the percentage of incorporated fluorescent aggregate into the crystals was monitored utilizing the fluorescent LMM and ground-based fluorescent microscopes.

Tracing transport of protein aggregates in microgravity versus unit gravity crystallization

“…The reduction in growth rate under microgravity versus unit gravity can be explained by the driving force ratio of diffusion as defined by Tanaka et al 47 , 48 . Data from SPX10 as well as previous studies demonstrated differences in the growth rate and geometry of crystals grown in microgravity versus unit gravity control experiments 44 , 45 , 49 . However, for the crystals under investigation, as mentioned previously, changes were not observed for shape and geometry for microgravity versus unit gravity crystals.…”

“…Our investigations addressed two prevailing theories regarding why microgravity-grown protein crystals often exhibit improved X-ray diffraction statistics compared to unit gravity control crystals. The first theory concerning differences in crystal growth rates found in our experiments was previously addressed 44 , 45 . The reduction in growth rate under microgravity versus unit gravity can be explained by the driving force ratio of diffusion as defined by Tanaka et al 47 , 48 .…”

“…A detailed statistical comparison of growth rates and crystal sizes along the different growth axes and in different sectors of the capillaries for microgravity and unit gravity crystallization was presented previously 44 , 45 and therefore is not the focus of this publication. In summary, it was found that the average growth rate of the major axis of needle-shaped Pf GST crystals and lysozyme crystals was found to be higher ( p < 0.01) at unit gravity compared to the microgravity environment.…”

On the Quality of Protein Crystals Grown under Diffusion Mass-transport Controlled Regime (I)

“…Consequently, it is not surprising that crystallization in space has received much financial support from both public space agencies and private companies until the last decade [1]. Since the early protein crystallization space adventure in 1984, many instruments have been tested under microgravity conditions [2][3][4][5][6][7]. At first, the results were not very conclusive, but interest did not decline, leading to a vast amount of research into reduced gravity environments [2][3][4][5][6][7], most of which reported crystal quality enhancements and strongly contributed to a better understanding of the nucleation and growth of protein crystals [4,[8][9][10].…”

“…Since the early protein crystallization space adventure in 1984, many instruments have been tested under microgravity conditions [2][3][4][5][6][7]. At first, the results were not very conclusive, but interest did not decline, leading to a vast amount of research into reduced gravity environments [2][3][4][5][6][7], most of which reported crystal quality enhancements and strongly contributed to a better understanding of the nucleation and growth of protein crystals [4,[8][9][10]. Although several parameters, such as the isoelectric point and water content, were not considered when analyzing the results, the observed improvement in crystal quality was explained on the basis of reduced crystal sedimentation, density-driven convection flows provoking the formation of depletion zones around growing crystals [11][12][13][14], and the reduced incorporation of impurities [4,15,16].…”