Crystallization of Medically Relevant Proteins Using Microgravity (Protein Crystallography) - 09.17.14
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Crystallizing proteins can help scientists determine the proteins’ atomic structure, which is important for developing new drugs as well as understanding how enzymes and other proteins function in the human body. In general, protein crystals grown in microgravity environments are larger and more perfect than crystals grown on Earth, where gravity interferes with the process. Crystallization of Medically Relevant Proteins Using Microgravity (Protein Crystallography) uses crystallization processes on the International Space Station to study the atomic structures of several key enzymes, which could be used to design new drugs to treat various diseases.
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OpNom CASIS PCG GCF-2
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
National Laboratory (NL)
Earth Benefits, Scientific Discovery
ISS Expedition Duration
March 2014 - September 2014
Previous ISS Missions
1) A ubiquitously expressed Ca2+-independent phospholipase A2 (group 6A, also called iPLA2β) is involved into multiple cellular functions and has a strong association with human diseases. It has a unique multidomain molecular structure, complex regulation and distinct physiological and pathological roles. There are very few tools to dissect the physiological functions of PLA2g6 in different tissues and there are no pharmacologically relevant inhibitors of the protein. The crystal structure of PLA2g6 will revolutionize the entire field by overcoming major limitations in understanding the distinct properties, mechanisms of regulation and function of its major splice variants. It will significantly advance design of novel inhibitors instrumental in treatment of related diseases including Parkinson’s disease, cardiovascular diseases, muscular dystrophy, diabetes, cancer and others.
2) Among all the enzymes involved in the blood coagulation, clotting, cascade, thrombin is the most important pharmacological target. Once generated from its inactive form prothrombin, thrombin promotes platelet aggregation (where small, disc shaped blood cell fragments clump together) by cleaving PAR-1 and PAR-4, and stabilizes the haemostatic plug (an agent that controls blood flow) by converting the soluble fibrinogen into the insoluble fibrin. Thrombin also catalyzes the conversion of the zymogen, Protein C, into the mature enzyme, activated protein C, which is, so far, the most potent anticoagulant in vivo. During the last few decades, much has been learned on thrombin function and regulation, but considerably less is known about the structural features of its precursors. The proposed studies are aimed to enhance researchers understanding of the molecular properties of prothrombin and its activation pathway to an entirely new level of detail that will eventually benefit and/or promote new strategies of therapeutic intervention.
Grow high resolution diffraction quality crystals of PLA2G6 and prothrombin using counter-diffusion technique in microgravity.
Novel structures of prothrombin and of phospholipase PLA2g6 will significantly advance understanding of fundamental molecular mechanisms implicated in cardiovascular, neurodegenerative and other diseases and will be highly instrumental in development of future treatments of related diseases including Parkinson’s disease, cardiovascular diseases, muscular dystrophy, diabetes, cancer and others.
The project includes 3 phases: 1) preparation of proteins for crystallization and optimization of crystallization conditions using counterdiffusion method in a capillary system identical to that of Granada Crystallization Facility; 2) preparation and loading solutions into GCF to turnover to space carrier; 3) collecting GCF and samples from GCBs after flight completion, performing X-ray diffraction experiments to collect diffraction data and solving tertiary structures of proteins using data collected from crystals grown in a microgravity environment. In step 2), parallel crystallization experiments are conducted on the ground in researcher’s labs for comparison purpose to compare crystals obtained under microgravity conditions to those performed under conventional conditions. All results will be published in peer-reviewed journals and presented at international meetings.
The majority of efforts focus on step 1. Both proteins require significant efforts and resources to be produced in an amount sufficient for crystallization experiments. Clones of C. griseus (CHO) PLA2G6 and human PLA2G6 containing a C-terminal 6xHis-tag are expressed in baculovirus-infected SF9 cells and purified using NiNTA and ATP-(or CaM-)agarose columns to more than 95% purity with yields between 1 to 4 mgs per 1 L of cell culture. The crystals belong to the hexameric space group with the dimensions of a=b=271 Å, c=80 Å. Crystals are characterized by an anisotropic diffraction from 4 to 6 Å resolution. The best crystals are characterized by a mosaicity as low as 0.4˚C and with an R-merge of 8% for 40-6 Å data range. Resolution of 3.2 Å can be achieved with an increased X-ray exposure; however, crystals quickly decay. Co-crystallization experiments with several non-cleavable substrates yielded small crystals with an altered morphology. Preliminary analysis of low-resolution diffraction revealed a similar space group and an isomorophous difference of 20-25% with data collected from crystals of apo-protein.
Prothrombin has been purified by flash frozen plasma. The chemical identity and purity were verified by SDS-PAGE and LC-MS. Gla-domainless prothrombin was produced by limited proteolysis and purified by size exclusion chromatography. Recombinant human full-length and Gla-domainless prothrombin have been cloned and expressed in mammalian cells and purified to homogeneity. Currently, researchers can obtain 3-4 mg of each protein construct per liter of media. For large-scale production of recombinant protein, cells were grown in cell factory system, the medium was harvested every 48 h and stored at 4˚C in 10 mM benzamidine. X-ray crystal structure of the Gla-domainless prothrombin solved at 3.2 Å resolution with a final Rfree=0.379. The Rfree is still significantly high since the crystal diffracts at 3.2-3.3 Å in one orientation but only at 5.5 Å in another orientation. The structure was solved by molecular replacement using prethrombin-2 mutant S195A (3SQH) as a search model. Kringle 2 was obtained from the structure of meizothrombin desF1 (3E6P). However, the electron density of the kringle 1 is very weak and its orientation/structure might dramatically change in a higher resolution dataset. Initial screening of recombinant version of the Gla-domainless prothrombin expressed in mammalian cells allowed researchers to obtain small crystals with a maximum resolution of 3.3 Å. While space group is identical, cell parameters are different, e.g. "b” axis value drops from 103.1 Å in form I to 94.0 Å in form III. There are two molecules in the asymmetric unit and they are significantly different. Scientists were able to identify all three domains in one molecule whereas in the second molecule the kringle 1 is missing. Whether the lack of the kringle 1 is due to crystal packing/organization, intrinsic disorder of this domain or other reasons is currently under investigation. This crystal form can potentially yield two alternative conformation of the same protein construct, providing essential mechanistic insight into domain flexibility in prothrombin structure.
Since both proteins were crystallized in a vapor diffusion system, crystallization conditions can be optimized to reproducibly obtain crystals in capillaries using Granada Crystallization Boxes. Since the experiment in phase 2 during flight is completely passive, it is important to optimize parameters of GCB to initiate the crystallization process after 12-24 hours after packing in order to start crystallization process when the carrier reaches the orbit. This can be achieved by optimizing thickness of agarose gel separating the protein solution in capillaries from the buffer containing precipitant.
After completion of the flight, the GCF is collected and protein crystals are extracted from the capillaries and subjected to a traditional data collection and structure solution analysis. Both structures are solved using molecular replacement techniques using structures of homologous domains.
Protein Crystallography builds upon previous research to improve methods for crystalizing proteins in microgravity. Researchers aim to obtain new data about protein crystals that are difficult to obtain under normal gravity conditions. Additionally, the investigation embodies the use of the ISS as a platform for scientific discovery.
Prothrombin is an enzyme involved in blood clotting and blood thinning in the body, and phospholipase PLA2g6 is an enzyme involved in a wide range of human diseases. Understanding the physical structure of prothrombin could help researchers develop new drugs for cardiovascular diseases. Understanding the structure of phospholipase PLA2g6 will be instrumental in developing future treatments for a variety of diseases, including heart disease, Parkinson’s disease, muscular dystrophy, diabetes, and cancer.
Experiment is completely passive and should remain undisturbed for entire mission until the return of the Space X Dragon Capsule. Experiment is activated in pre-launch loading but onset of crystallization is delayed by a gel which slows the diffusions of the sample.
No on-orbit operations.