Effect of macromolecular mass transport in microgravity protein crystallization

Abstract

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.