Protein Crystals for Neutron Crystallography (PC4NC) Large volume crystal growth of Inorganic Pyrophosphatase complexes by counter-diffusion in microgravity for neutron diffraction studies (PC4NC) - 07.14.16
Enzymes play a critical role in many biological processes, but the way they operate is not well understood. Protein Crystals for Neutron Crystallography (PC4NC) studies an enzyme called inorganic pyrophosphatase (IPPase) to determine how it functions. By studying the diffraction of neutrons in the crystal, researchers can locate the positions of hydrogen bonds in the enzyme, which will help determine how the molecule works in a cell. In order to do so, large enzyme crystals must be grown, but this is only possible in the microgravity environment of the International Space Station. Science Results for Everyone
Information Pending Experiment Details
Joseph D. Ng, Ph.D., iXpressGenes, Hunstville, AL, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
National Laboratory (NL)
Earth Benefits, Scientific Discovery
ISS Expedition Duration
March 2014 - September 2014
- Complexes of the inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens are the targets for large volume crystal (1mm3) growth for Neutron Macromolecular Crystallography (NMC). Counter- diffusion crystallization in capillaries having diameters of 1mm or greater are used in the Granada Box Facility to achieve this goal. A microgravity environment is essential to form a stable supersaturation gradient to obtain these large crystals. The principal outcome is to identify the role of H atoms in enzymatic activity and decipher a structure-based mechanism for IPPase and its complexes where this would not be possible otherwise with previous crystallographic models determined from Earth-grown crystals.
To grow protein crystals in large-diameter capillaries in a diffusion limiting mass transport process on Earth and under microgravity.
- IPPase complexes- over 25 different protein form IPPases is prepared to be crystallized by counter-diffusion in restricted geometry. The crystal growth process is observed and measured to further understand its equilibration process in different configurations.
- D2O uptake by the crystal in the restricted geometry format is measured to quantify deuterium exchange rates and optimize D2O exchange protocols.
- Perdeuterated IPPase is also crystallized in the same format to observe crystal growth differences due to isotope effects.
To determine the total protein structures of IPPase and other hyperthermophilic proteins by Neutron Crystallography.
- Initial neutron diffraction analysis is performed on single large protein crystals for preliminary evaluation.
- Complete neutron crystallographic data collection for selected proteins grown on Earth and those that have been obtained under microgravity are performed.
- Neutron crystallographic data sets are coupled to already existing X-ray crystallographic structures for total protein structure determination. Since neutron diffraction does not cause any radiation damage, protein crystals that have been analyzed by NMC are also evaluated by synchrotron X-ray.
To obtain mechanistic models of protein catalysis and binding for selected proteins.
- Determine hydrogen positions and protonation states of IPPases complexed to different metals, pyrophosphate substrates and inhibitors from neutron and X-ray diffraction data.
- Biochemical characterization is performed involving kinetic and binding measurements to accompany structural data.
- Propose a comprehensive molecular model of catalysis.
Unlike X-ray diffraction, which can also unveil the structure of a material, neutron diffraction requires large volumes of crystals, which are difficult to grow on Earth. Microgravity is an ideal environment for crystallizing large molecules like proteins, because gravity does not affect how the crystal structures form. More than 80,000 protein structures have been derived using X-ray diffraction, while only about 50 have been studied with neutron diffraction. The International Space Station may be an essential environment to obtain protein crystals suitable for neutron diffraction studies.
Successfully determining the structure of IPPase would be a major breakthrough for medical science on Earth. Crystallizing the enzyme would reveal its mechanisms for the first time, allowing researchers to understand the atomic reactions involved in this type of transfer enzyme. Understanding how this enzyme works could enable the design of new antibiotics. It could also improve processing for a common type of DNA sequencing technology known as polymerase chain reaction, projected to be worth $27 billion by 2015.
Operational Requirements and Protocols
Decadal Survey Recommendations
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