Crystallization of the collagen-like polypeptide (PPG)₁₀ aboard the International Space Station. 1. Video observation

Abstract

Single chains of the collagen model polypeptide with sequence (Pro-Pro-Gly)(10), hereafter referred to as (PPG)(10), aggregate to form rod-shaped triple helices. Crystals of (PPG)(10) were grown in the Advanced Protein Crystallization Facility (APCF) both onboard the International Space Station (ISS) and on Earth. The experiments allow the direct comparison of four different crystallization environments for the first time: solution in microgravity ((g), agarose gel in (g, solution on earth, and gel on earth. Both on board and on ground, the crystal growth was monitored by a CCD video camera. The image analysis provided information on the spatial distribution of the crystals, their movement and their growth rate. The analysis of the distribution of crystals reveals that the crystallization process occurs as it does in batch conditions. Slow motions have been observed onboard the ISS. Different to Space-Shuttle experiment, the crystals onboard the ISS moved coherently and followed parallel trajectories. Growth rate and induction time are very similar both in gel and in solution, suggesting that the crystal growth rate is controlled by the kinetics at the interface under the used experimental conditions. These results provide the first data in the crystallogenesis of (PPG)(10), which is a representative member of non-globular, rod-like proteins.

Phase behavior and crystallogenesis under counter-diffusion conditions of the collagen-model peptide (Pro–Pro–Gly)10

“…Keeping the capillaries in horizontal position (i.e. perpendicular to the gravity vector) reduces the motion of crystals in the on-ground experiments, as reported in the APCF reactors [21,22]. The morphology of crystals grown in solution and gel, both in mg and on ground, was slightly different.…”

“…1b. (PPG) 10 solubility decreases at increasing PEG weight fraction, w i (v/v for PEG400 and w/v for higher molecular mass PEGs), for all samples, but with different slopes P ( P =À(qlogS/qw i ) T ). Although the isotropic-nematic coexistence region should broaden on adding PEG, no liquid-liquid phase separation was observed in these solutions, but it was macroscopically observed in an APCF reactor [28], where (PPG) 10 was crystallized with PEG400 (10%v/v) in agarose gel (0.2% w/v), both on ground and in mg [22] (Supplementary material). Perhaps, the liquid-liquid phase transition does not occur at equilibrium, but does occur between two metastable liquid phases, stabilized by the convection-free agarose gel matrix.…”

“…Being (PPG) 10 charged at both the C-and N-term, the effect of the pH is presumably associated to a strong head-tail electrostatic interaction, quantitatively explained according to a reliable isoelectric point of 5.370.1, that corresponds to the solubility minimum. This is exactly the optimal pH at which (PPG) 10 high-quality crystals (diffracting up to 1.3 Å ) were obtained by a trial and error procedure [22]. This value was kept also in the crystallogenic work herein reported in order to perform an accurate comparison.…”

“…Therefore, we used an initial supersaturation value of 14-30 (evaluated as a ratio between initial protein concentration and solubility data). Density of the solutions were the same as previously published [22,23]. On ground, capillaries were kept in a horizontal position (x-y plane) that avoids sedimentation along the z-direction.…”

“…This paper provides new results to this debate. We herein compare the results of a Space Mission on board the International Space Station (ISS), in which the Granada Crystallization Facility (GCF) [18], was used, with results previously obtained using the Advanced Protein Crystallization Facility [19], APCF (now upgraded to PCDF [20,21]) onboard the ISS [22]. We have designed our crystallization experiments of (PPG) 10 in mg to compare crystallogenic and crystallographic outcomes from different crystallization facilities.…”