Microgravity Crystal Growth for Improvement in Neutron Diffraction and the Analysis of Protein Complexes (CASIS PCG 8) - 06.20.18

Overview | Description | Applications | Operations | Results | Publications | Imagery

ISS Science for Everyone

Science Objectives for Everyone
Microgravity Crystal Growth for Improvement in Neutron Diffraction and the Analysis of Protein Complexes (CASIS PCG 8) plans to grow large, high-quality crystals of two Vitamin B6-dependent enzymes, porcine cytosolic aspartate aminotransferase (AAT) and Salmonella typhimurium tryptophan synthase Type 1 (TS) and of a transient deoxyribonucleic acid (DNA) repair complex (interactions between two proteins) obtained from a bacteriophage (RNH:32). Neutron diffraction studies of the crystals are conducted at Oak Ridge National Laboratories to better understand catalyst mechanisms and structures, particularly positions of hydrogen atoms which are visible to neutron but not X-rays. In the second study, the transient and flexible interaction between two DNA repair proteins is visualized. Crystals grown in microgravity are less flexibly since thermal convections are absent; this makes their structures more uniform and the attained information is at higher resolution.
Science Results for Everyone
Information Pending

The following content was provided by Timothy Mueser, Ph.D., and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: PCG-8

Principal Investigator(s)
Timothy Mueser, Ph.D., University of Toledo, Toledo, OH, United States

Co-Investigator(s)/Collaborator(s)
Information Pending

Developer(s)
Center for the Advancement of Science in Space (CASIS), Melbourne, FL, United States
The Bionetics Corporation, Yorktown, VA, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Information Pending

ISS Expedition Duration
February 2018 - August 2018

Expeditions Assigned
55/56

Previous Missions
SpX-4

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Experiment Description

Research Overview

  • The information obtained from Microgravity Crystal Growth for Improvement in Neutron Diffraction and the Analysis of Protein Complexes (CASIS PCG 8) may allow: 1.) the comparison of two reaction mechanisms in Pyridoxal Phosphate (PLP)-dependent enzymes to discern differences in mechanism, and 2.) the visualization of a transient interaction in DNA repair.
  • The use of liquid/liquid diffusion in microgravity improves the size of protein crystals which is essential for neutron diffraction analysis.
  • The lack of buoyancy and sedimentation in microgravity is expected to result in dramatic improvement in crystal quality.
  • A workflow between two national laboratories is being established while using these model proteins. That is, the use of microgravity crystal growth aboard the International Space Station (ISS) is being paired with the neutron diffraction studies of macromolecules at Oak Ridge National Laboratory (ORNL).

Description

Microgravity Crystal Growth for Improvement in Neutron Diffraction and the Analysis of Protein Complexes (CASIS PCG 8) utilizes microgravity to improve the size and quality of protein crystals for neutron and X-ray diffraction analysis. Protein crystals grow from conditions that are allowed to change gradually over time. For these experiments, glass capillaries are filled with protein solution and placed in a crystallization solution that enters the capillary through a barrier placed at one end, a technique known as liquid/liquid diffusion. This technique is being used in two studies: 1.) to improve the size and quality of the protein crystals of pyridoxal phosphate-dependent enzymes for neutron diffraction analysis, and 2.) to improve the crystal quality of protein complexes that are not inherently stable but instead form transiently during a reaction.
 
Pyridoxal phosphate (PLP) is an enzyme cofactor derived from Vitamin B6. PLP is the most common vitamin cofactor and is involved in approximately 6% of all metabolic reactions. The most intriguing aspect is the wide variety of reactions in which PLP is employed by nature. Porcine cytosolic aspartate aminotransferase (AAT) and Salmonella typhimurium tryptophan synthase Type 1 (TS) are the two model PLP-dependent enzymes. Both enzymes use PLP to couple to an amino acid in an identical fashion, but the products formed are very different. For AAT, the amino acid is deaminated yielding a ketoacid. For tryptophan synthase, the substrate undergoes a beta elimination, creating an intermediate required for coupling serine to indole to form tryptophan.
 
The differences in catalytic outcomes of the PLP enzymes are dependent on protonation state of the cofactor. X-ray diffraction is used to locate the heavier atoms – carbon, nitrogen, and oxygen – in protein crystals. Hydrogens, which are nearly half of the atoms in a protein, are essentially invisible to X-rays; therefore, Neutron diffraction is used to locate the position of deuterium, an isotope of hydrogen. The diffraction signal of neutrons scattered by deuterium is comparable to the heavier atoms. Joint refinement using X-ray and neutrons allows positioning of all atoms in the protein structure. The difficulty lies in the fact that neutron sources are low intensity compared to X-ray sources. The crystals used in neutron diffraction require a much larger sample size, 2 to 3 orders of magnitude larger in volume. AAT and TS are being subjected to microgravity conditions to improve the size and quality of the crystals for neutron diffraction studies.
 
Deoxyribonucleic acid (DNA) is the only macromolecule with dedicated repair. Progeny cells are insured to inherit damage-free information. The flap endonucleases (FEN) are a family of enzymes that recognize and remove short strands of DNA displaced by synthesis. Recognition is structure-specific and repair is assisted by other factors. Most protein complexes are stable and appear in a specific form in crystals. The interaction of T4 RNaseH (RNH), a FEN enzyme from a bacteriophage, and the assisting factor, GP32, a single-stranded DNA-binding protein, is transient and flexible. The two proteins form a stable binary complex in solution but the flexibility of the complex creates defects in the crystals referred to as mosaicity. Placing the binary complex in microgravity mitigates the gravity-based convections that contribute to the high crystal mosaicity. Using the liquid/liquid diffusion method, the crystals of the RNH:GP32 complex formed in microgravity are expected to have higher uniformity and display higher resolution in X-ray diffraction experiments.

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Applications

Space Applications
On Earth, crystal defects occur that affect the quality of crystals used in neutron and X-ray diffraction data. Larger, higher-quality crystals grow in microgravity due to the lack of thermal gradients affecting convection.

Earth Applications
Humans lack the biosynthetic pathway for tryptophan, an essential amino acid, whose pathway is found in a microbe. Cytosolic aspartate aminotransferase is a biomarker used to diagnose a heart attack. Improvement of crystals of transient complexes may enable considerable advancement in all areas employing structural biology.

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Operations

Operational Requirements and Protocols
The hardware consists of small glass capillaries in polyethylene bags. The capillaries contain the protein and the precipitant solution is in the polyethylene bags. The precipitant solution slowly diffuses through an agar plug in the capillary so that nucleation and crystal growth occur in microgravity. A total of 120 samples are flown, spread out amongst three plastic boxes. The boxes provide a rigid structure for the capillaries and bags, preventing damage. The experiment launches at refrigerated (+4°C) temperatures and transfers to refrigerated storage aboard the ISS. The crew members take photos of the hardware during the transfer procedures. The experiment also returns refrigerated.

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Decadal Survey Recommendations

Information Pending

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Results/More Information

Information Pending

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Related Websites
The University of Toledo, Department of Chemistry and Biochemistry

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Imagery