Optimization of Protein Crystal Growth for Determination of Enzyme Mechanisms through Advanced Diffraction Techniques (Protein Crystal Optimization ) - 05.13.15

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Optimization of Protein Crystal Growth for Determination of Enzyme Mechanisms through Advanced Diffraction Techniques (Protein Crystal Optimization) produces large, high-quality protein crystals that can be used to study how enzymes work. Neutron diffraction crystallography can distinguish individual protons and atoms, but it requires large perfect crystals, which are difficult or impossible to make under the influence of gravity on Earth. The International Space Station's microgravity environment is ideal for testing how well scientists can crystallize certain proteins that are important for medical science.
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The following content was provided by Constance Schall, and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: CASIS PCG2

Principal Investigator(s)
Constance Schall, Department of Chemical and Environmental Engineering, Toledo, OH, United States

Co-Investigator(s)/Collaborator(s)
Timothy Mueser, University of Toledo, Toledo, OH, United States
Donald Ronning, University of Toledo, Toledo, OH, United States
Leif B. Hanson, University of Toledo, Toledo, OH, United States
Julian Chen, University of Toledo, Toledo, OH, United States
Paul Langan, Biology &Soft Matter Div, Oak Ridge, TN, United States
Andrey Kovalevsky, Oak Ridge National Laboratory, Oak Ridge, TN, United States
Zoe S. Fisher, Los Alamos National Laboratory, Los Alamos, NM, United States

Developer(s)
Center for the Advancement of Science in Space (CASIS), Rockledge, FL, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Information Pending

ISS Expedition Duration
March 2014 - September 2014

Expeditions Assigned
39/40

Previous ISS Missions
Information Pending

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Experiment Description

Research Overview

  • To date, many earth grown crystals have not been of high enough quality to fully model using neutron diffraction crystallography. With the absence of gravity and convection on the International Space Station, larger crystals with purer compositions and fewer defects can be grown.
  • Single neutron and X-ray diffraction quality crystals are grown, inspected and characterized by similar traits.
  • The target proteins have large potential in novel drug development in healthcare. One of the proteins also has potential application as a target for control of Salmonella in the food and beverage industries.

Description
This investigation seeks to exploit the microgravity environment to improve the quality and yield of protein crystals. The crystals of three medically relevant proteins are used for neutron diffraction experiments that can directly lead to determination of the neutron crystal structures. To date, neutron structures of these proteins have not yet been determined with terrestrially grown crystals, as crystals of these proteins contain defects that can only be forestalled by growth in microgravity. Ancillary ground-based studies are also completed to improve and refine the growth conditions used in microgravity. 

 

Half of all atoms in a protein are hydrogen, which play critical roles in hydrogen bonding and enzyme mechanisms. Although X-ray diffraction has had unparalleled success in detailing macromolecular structure, hydrogen atoms scatter X-rays weakly, and only a limited number are visible even in the highest resolution structures. In contrast, hydrogen isotopes (H and D) scatter neutrons in a manner such that they can be readily visualized in neutron diffraction structures even at modest resolution. However, because of the weak interaction of neutrons with matter and the relatively weak flux from neutron sources, neutron diffraction studies require larger crystals than X-ray diffraction. The benefits of microgravity crystal growth are therefore especially relevant to neutron diffraction, with the potential to grow larger, more perfect crystals, especially from proteins that do not reach acceptable size or quality at unit gravity.

 

Targeting multidrug-resistant bacterial strains is one of the most challenging issues in modern medicine. One way to facilitate the development of new broad-spectrum antibiotics is to target enzymes involved in multiple bacterial biosynthetic pathways. One such pathway is quorum sensing the cell density-dependent communication system of bacteria. Quorum sensing systems regulate pathogen –host interactions, bacterial virulence, and the formation of bacterial biofilms. One quorum sensing molecule, autoinducing molecule (AI)2, is used by both Gram-positive and Gram-negative bacteria and is derived from S-adenosylhomocysteine (SAH). At the nexus of autoinducer biosynthesis, menaquinone biosynthesis, and SAH metabolism is 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN). A multi-functional bacterial enzyme, MTAN catalyzes the hydrolysis of the N-glycosidic bond of many adenosine-based metabolites. ‘Snapshots’ of important steps in the MTAN reaction mechanism can be obtained through X-ray structures of MTAN complexes with substrate, product , and substrate analogs such Formycin A (FMA). Subsequent data sets yielded high resolution structures, but a number of solvent networks, hydrogen-bonded networks,  and protonation states of key residues could not be elucidated, hence the desire to use neutron diffraction studies to clarify the reaction mechanism of MTAN. It is expected that X-ray and neutron structures will lay the groundwork for drug design efforts targeting this essential bacterial enzyme.

 

Vitamin B6 and its biologically active phosphorylated derivative pyridoxal-5’-phosphate (PLP) are among the most versatile cofactors used in nature. Because enzymes dependent on vitamin B6 or PLP are ubiquitous and serve important roles in amino acid and glycogen metabolism, they are targets for the design of specific inhibitors, both for medical and public safety purposes. To better understand PLP cofactor-assisted catalysis and to facilitate ligand design, the investigator will attempt neutron diffraction studies of Salmonella typhimurium tryptophan synthase (TS) and porcine cytosolic aspartate aminotransferase (AAT). TS catalyzes the reaction of indole with serine to form tryptophan. TS has no human or animal counterpart, making it an attractive target for broad-spectrum inhibition of pathogenic organisms. Like MTAN, crystal defects such as solvent inclusions and anisotropic growth are apparent at unit gravity. The suppression of buoyancy driven convection and sedimentation in microgravity is expected to result in dramatic improvement in crystal quality.

 

AAT is one of the key enzymes in amino acid metabolism. AAT is also one of the primary biomarkers for liver and heart disease. Human and porcine AAT share 94% identity. However, porcine protein more readily forms larger crystals that diffract to higher resolution. Crystals of porcine AAT can be grown to a large size on earth, but contain numerous defects that make them so far unsuitable for neutron diffraction studies. 

 

The project uses very few resources and has a flexible strategy that makes it an ideal system for ISS. Two Handheld HDPCG hardware units launch in cold stowage hardware at +22°C and at +4°C, respectively. The +22°C unit transfers to ISS ambient stowage and the +4°C unit transfers to refrigerated storage on ISS. The experiment is activated shortly after arrival to ISS by turning the sample cells 90° clockwise to introduce the sample insert to the precipitant reservoir. Following approximately 26 days of crystal growth in the ambient unit and up to 6 months in the refrigerated unit, the sample cells are turned 90° counter-clockwise to turn the protein insert opposite of the precipitant reservoir. The ambient unit returns at ambient in cold stowage hardware and the refrigerated unit returns at +4°C in cold stowage hardware. 

 

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Applications

Space Applications
Neutron diffraction is a method of locating individual hydrogen atoms in a three-dimensional crystallized form of a molecule. This provides key insight into how the molecule functions. For proteins and enzymes involved in human health and disease, understanding molecular mechanisms can help scientists design better drugs. But on Earth, gravity interferes with the growth of 3-D molecular crystals, so this investigation depends on the unique microgravity environment of the ISS. Data from crystal growth also adds to the growing body of research on crystal optimization in microgravity, and demonstrates the benefits of space research to people on Earth.

Earth Applications
The investigation studies three target proteins that hold promise for new therapies for cancer, drug-resistant bacterial infections, and chronic disease. MtaN is an important drug target for the development of new drugs to treat infections by drug-resistant bacteria such as Helicobacter pylori (Hp), which causes ulcers and may be related to gastric cancer.  TS (typhimurium tryptophan synthase) has potential for control of salmonella bacteria in the food industry. And AAT is a biomarker for heart and liver disease. High-quality crystals of these proteins enable a better understanding of how they work, which improves the process of drug discovery and development.

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Operations

Operational Requirements

  • Time between turnover and launch: L-24 hours

    • 1 HDPCG at +22°C ambient cold stowage

    • 1 HDPCG at +4°C in cold stowage

  • Scrub turnaround requirement: 48 hours from initial T-0

  • Launch: turnover temperatures maintained until crew access on ISS

  • On-orbit:

    • 1 HDPCG at ambient on ISS, 1 HDPCG maintained at +4°C while on ISS

    • Sample activation must be completed within six days following launch

    • Sample deactivation (ambient HDPCG) must occur as close to undock as possible to ensure a minimum experiment duration of 23 days 

  • SpX-4 Return: 1 ambient HDPCG

    • Ambient temperature range (+18°-25°C) must be maintained during return

    • Early recovery at the LA area airport required, samples will be shipped to laboratory for neutron diffraction studies

  • SpX-5 Return: 1 +4°C HDPCG

    • Temperature cannot fall below +1°C or rise above +6°C

Operational Protocols
Two Handheld HDPCG hardware units are flown, one at ambient (+22°C) and one refrigerated (+4°C). Both units are activated during removal from the cold stowage hardware. To activate the experiment, an Activation Tool (attached to the side of the HDPCG hardware using Velcro) is removed and attached to each cell block (5 total per HDPCG unit). The cell block is turned 90° clockwise to align the sample insert with the precipitant solution. Crystals in the ambient unit grow for up to 28 days and are then deactivated by attaching the Activation Tool to each cell block and turning 90° counter-clockwise to turn the protein insert opposite of the precipitant reservoir. The ambient HDPCG unit is then transferred to ambient cold stowage hardware for return. Crystals in the refrigerated HDPCG unit grow for up to 6 months and are then deactivated using the same procedure as the ambient unit. The refrigerated HDPCG unit is then transferred to +4°C cold stowage hardware for return. 

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Results/More Information

Information Pending

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Related Websites
The University of Toledo
CASIS
The University of Toledo Department of Chemistry

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Imagery