Perfect Crystals: microgravity capillary counterdiffusion crystallization of human manganese superoxide dismutase for neutron crystallography
Year: 2023
Type: article
Abstract: The NASA mission Perfect Crystals used the microgravity environment on the International Space Station (ISS) to grow crystals of human manganese superoxide dismutase (MnSOD)-an oxidoreductase critical for mitochondrial vitality and human health. The mission's overarching aim is to perform neutron protein crystallography (NPC) on MnSOD to directly visualize proton positions and derive a chemical understanding of the concerted proton electron transfers performed by the enzyme. Large crystals that are perfect enough to diffract neutrons to sufficient resolution are essential for NPC. This combination, large and perfect, is hard to achieve on Earth due to gravity-induced convective mixing. Capillary counterdiffusion methods were developed that provided a gradient of conditions for crystal growth along with a built-in time delay that prevented premature crystallization before stowage on the ISS. Here, we report a highly successful and versatile crystallization system to grow a plethora of crystals for high-resolution NPC.
Source: npj Microgravity
Authors: William E. Lutz, Jahaun Azadmanesh, Jeffrey J. Lovelace, Carol Kolar, Leighton Coates, Kevin L. Weiss, Gloria E. O. Borgstahl
Language: English
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $3,290
Funders: U.S. Department of Energy, U.S. Department of Health and Human Services, National Aeronautics and Space Administration, Nebraska Space Grant Consortium, University of Dundee, Imperial College London, Commonwealth Scientific and Industrial Research Organisation, Nuclear Safety and Security Commission, Universidad de Granada, National Institutes of Health, Office of Science, Kennedy Space Center, Marshall Space Flight Center, Johnson Space Center, National Cancer Institute, National Institute of General Medical Sciences, Basic Energy Sciences, Biological and Environmental Research, Office of Experimental Program to Stimulate Competitive Research, Oak Ridge National Laboratory
Awards: G164730967, G1663022414, SUPEROXIDE DISMUTASES (SODS) ARE IMPORTANT ANTIOXIDANT ENZYMES THAT PROTECT ALL MVING CELLS AGAINST TOXIC OXYGEN METABOLITES, ALSO KNOWN AS REACTIVE, G3418376741, G4423567074, Computational Tools for Neutron Protein Crystallography, SUPEROXIDE DISMUTASES (SODS) ARE IMPORTANT ANTIOXIDANT ENZYMES THAT PROTECT ALL LIVING CELLS AGAINST TOXIC OXYGEN METABOLITES ALSO KNOWN AS REACTIVE OXYGEN SPECIES (ROS). SODS ARE THE FIRST DEFENSE AGAINST PROPAGATION OF DAMAGING OXIDATIVE REACTIONS THROUGH ELIMINATION OF SUPEROXIDE. SUPEROXIDE IS GENERATED THROUGH NORMAL METABOLISM AND/OR IONIZING RADIATION. EACH CATALYTIC CYCLE DISMUTES TWO MOLECULES OF SUPEROXIDE TO OXYGEN AND HYDROGEN PEROXIDE VIA CYCLIC REDUCTION AND OXIDATION HALF REACTIONS USING THE ACTIVE SITE METAL ION. HUMANS HAVE CU/ZNSOD IN THE CYTOSOL AND EXTRACELLULAR SPACES AND MNSOD IN THEIR MITOCHONDRIA. MUTATIONS IN SOD LEAD TO AGING AND DEGENERATIVE DISEASES SUCH AS AMYOTROPHIC LATERAL SCLEROSIS DIABETES AND CANCER. THIS FLIGHT PROPOSAL WILL PROVIDE THE CRITICAL CRYSTAL SAMPLES NEEDED FOR A DETAILED STUDY OF HUMAN SOD. DESPITE THE BIOLOGICAL AND MEDICAL IMPORTANCE OF SOD THE COMPLETE ENZYMATIC MECHANISM IS STILL UNKNOWN. PRECISE STRUCTURAL DATA ARE NEEDED. THE BINDING SITES OF THE DIATOMIC SUBSTRATE AND PRODUCT AS WELL AS THE SOURCE OF THE PROTONS IN THE REACTION HAVE BEEN STUDIED BUT THEIR EXACT IDENTIFICATION HAS NOT BEEN POSSIBLE. THIS DETAILED INFORMATION CAN ONLY BE DETERMINED BY NEUTRON DIFFRACTION. COMPLEXES OF HUMAN MNSOD INCLUDING STRUCTURAL INTERMEDIATES AND MUTANTS WILL BE THE TARGETS FOR LARGE VOLUME CRYSTAL ( 1MM3) GROWTH FOR STRUCTURE DETERMINATION BY NEUTRON MACROMOLECULAR CRYSTALLOGRAPHY (NMC). THE QUIESCENT ENVIRONMENT AFFORDED BY MICROGRAVITY IS KNOWN TO GROW CRYSTALS LARGE ENOUGH FOR NEUTRON STUDIES NOT ONLY ARE THEY LARGE BUT THEIR QUALITY APPROACHES PERFECTION. IN 2001 THE BORGSTAHL LABORATORY SUCCESSFULLY GREW LARGE CRYSTALS OF SOD USING MICROGRAVITY CONDITIONS ON THE INTERNATIONAL SPACE STATION (ISS). WITH NASA S RENEWED INTEREST IN IMPLEMENTING THE MICROGRAVITY ENVIRONMENT ON THE ISS FOR PROTEIN CRYSTAL GROWTH WE WOULD LIKE TO MOVE FORWARD WITH THESE EXCITING EARLY MICROGRAVITY CRYSTALLIZATION RESULTS FOR SOD. EXISTING CRYSTALLIZATION HARDWARE THAT USES THE GRANADA CRYSTALLIZATION BOXES (GCB) FOR CAPILLARY COUNTERDIFFUSION PROTOCOLS WILL BE USED TO ACHIEVE THESE GOALS. A MICROGRAVITY ENVIRONMENT IS ESSENTIAL TO FORM A STABLE SUPERSATURATION GRADIENT TO OBTAIN THE LARGE HIGH QUALITY CRYSTALS REQUIRED FOR NMC. THEN NMC WILL BE PERFORMED WITH COLLABORATORS AT OAK RIDGE NATIONAL LABORATORY (ORNL). THE PRINCIPAL OUTCOME WILL BE TO IDENTIFY THE ROLE OF HYDROGEN ATOMS IN ENZYMATIC ACTIVITY DISCERN SUPEROXIDE FROM PEROXIDE AND WATER FROM HYDROXIDE ION BY THEIR PROTONATION STATE AND DECIPHER A STRUCTURE-BASED MECHANISM FOR HUMAN MNSOD MORE PRECISELY THAN FROM PREVIOUS X-RAY CRYSTALLOGRAPHIC MODELS DETERMINED FROM EARTH-GROWN CRYSTALS. THESE CONTRIBUTIONS WILL ALSO PROVIDE CRITERIA NEEDED FOR THE PROTEIN ENGINEERING OF DESIRABLE PROPERTIES INTO ENZYMATIC METAL CENTERS FOR PROTON COUPLED ELECTRON TRANSFER., G6554919817, G8880162300, Deciphering the Enzymatic Mechanism of Superoxide Dismutase, Buffett Cancer Center Support Grant
OpenAlex ID: W4379260574