The Effect of Macromolecular Transport of Microgravity Protein Crystallization (LMM Biophysics 1) - 02.08.17

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Proteins are important biological molecules that can be crystallized to provide better views of their structure, which helps scientists understand how they work. Proteins crystallized in microgravity are often higher quality than those grown on Earth. The Effect of Macromolecular Transport on Microgravity Protein Crystallization (LMM Biophysics 1) studies why this is the case, examining the movement of single protein molecules in microgravity.
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The following content was provided by Lawrence J. DeLucas, O.D., PhD, and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom:

Principal Investigator(s)
Lawrence J. DeLucas, O.D., PhD, University of Alabama, Birmingham, AL, United States

Co-Investigator(s)/Collaborator(s)
Christian Betzel, PhD, University of Hamburg, Germany

Developer(s)
ZIN Technologies Incorporated, Cleveland, OH, United States
NASA Glenn Research Center, Cleveland, OH, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Earth Benefits, Scientific Discovery

ISS Expedition Duration
March 2016 - February 2017; March 2017 - September 2017

Expeditions Assigned
47/48,49/50,51/52

Previous Missions
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Experiment Description

Research Overview

  • Proteins are important macromolecules without which our bodies would be unable to repair, regulate, or protect themselves. Researchers grow crystals from these proteins in order to determine their three dimensional structure. Using this 3-D structural information, researchers can determine how proteins function and which ones are involved in disease processes. The structure is often used to design new drugs that specifically interact with the protein. Many leading drugs today were discovered using their 3D structural information. While enormous strides have been made in the last decade, there still remain a large number of important proteins without structures due to the difficulty in growing the crystals needed to determine their atomic structure.
  • One class of proteins, membrane proteins, comprises a number of targets identified by the pharmaceutical industry as high-value commercial opportunities. Membrane protein crystal growth represents one area where the onset of space commercialization can maximize the impact of the microgravity environment in space-based research applications for the academic, government and pharmaceutical industries. Thus far only three membrane proteins have been crystallized in microgravity, yet results were promising for two of these: 1) a Photosynthetic Reaction Center-1 [photosystem-1], and 2) bacteriorhodopsin.
  • Access to unique data optimized in microgravity could have great relevance for understanding protein structures and advancing new drugs into the pharmaceutical market.
  • The Effect of Macromolecular Transport of Microgravity Protein Crystallization (LMM Biophysics 1) investigation looks at the underlying reason for the improved quality of microgravity-grown protein crystals. The present theories suggest that the improved quality of protein crystals grown in microgravity could be the result of two characteristics that exist in a buoyancy-free, diffusion-dominated solution. These are: 1) in microgravity the rate of protein transport is significantly decreased thereby causing crystals to grow at significantly slower rates. This characteristic enables the individual protein molecules to become more perfectly aligned in the crystalline lattice prior to additional protein molecules entering the lattice and interfering with alignment. 2) Predilection of growing crystals to incorporate protein monomers versus higher protein aggregates due to larger differences in transport rates in the diffusion dominated environment.

Description

Experimental Approach of the LMM Biophysics 1 Investigation:
Four aqueous proteins, one membrane protein (P-glycoprotein) and one virus (tobacco mosaic virus, TMV), are selected with the proteins covering a broad range of molecular weights (i.e. ~10kDa to as high as 1,250kDa) and tobacco mosaic virus (TMV) which exhibits a particle diameter of 18.0 nm and a length of ~300 nm. Purified batches of protein and virus particles are produced and isolated in different aggregate populations as outlined in the paragraphs below. Inclusion of P-glycoprotein (an integral membrane protein) and TMV is based on experience within the Center for Biophysical Sciences and Engineering (CBSE) expressing and purifying large quantities, as well as crystallizing this particular membrane protein and virus. Other potential protein candidates include cytochrome c (~12kDa), Glutathione–S-transferase (31kDa) and Thioredoxin (16kDa), α-chymotrypsinogen (25kDa), equine or bovine serum albumin (both ~66kDa), and Xenorhabdus-A, XptA1 (1,250kDa). The CBSE has significant experience expressing, purifying and producing crystals of each of these proteins. The final proteins selected are primarily based on the stability evaluation of the higher order aggregates of each protein. Dr. DeLucas’s laboratory has access to more than 100 different aqueous proteins covering the molecular weight range discussed above. Thus, additional candidates are available if needed.
 
Specific Aim 1 of LMM Biophysics 1 (aggregate incorporation into growing protein crystals):
As noted in the introduction section, purified proteins generally exist in solution with some small percentage of aggregates (i.e. dimers, trimers, tetramers, etc.). The LMM’s confocal fluorescent microscope is used to estimate the percent incorporation of aggregates into growing crystals of several different proteins.
 
Specific Aim 2 of LMM Biophysics 1 (comparison of µg versus 1-G crystal growth rates):
Specific Aim-2 is accomplished using the identical protein preparation methods as for specific aim-1. Once the growing crystals are identified via the microscope (or any LMM light microscope with 25x to 50x magnification), a series of images is taken over a period of 3 to 5 days to measure the crystal growth rates for each protein candidate. The growth rates for crystals grown in the microgravity environment are compared with 1-G control experiments for the same proteins. The effect of microgravity versus 1g on crystal growth rates is assessed for proteins of different molecular weights, and for protein solutions containing higher order protein aggregates. Solutions containing crystals are maintained in optical cells at either 4°C, or 22°C, and returned to earth for x-ray crystallographic analysis to assess crystal quality.
 
Specific Aim 3 of LMM Biophysics 1 (compare defect density/quality of crystals grown at different rates in 1g):
Dr. Christian Betzel (co-investigator from Univ. of Hamburg) recently developed a novel technology (Xtal ControllerTM) that provides a unique combination of diagnostic and control capabilities allowing real-time manipulation of the crystallization drop composition. Evaporation rates and crystal growth rates can be adjusted, thereby enabling a detailed comparison of crystal quality versus crystal growth rates. This unique crystallization system is utilized to achieve Specific Aim-3. Analysis of the quality of crystals grown at different rates is performed using atomic force microscopy [37, 38], and x-ray diffraction [11, 21]. The Xtal Controller is located in Dr. Betzel’s laboratory at the University of Hamburg. Both Dr. Betzel’s and Dr. DeLucas’ labs can use this unique system to precisely control key crystallization variables (i.e. protein concentration, temperature, precipitant concentration, additive concentrations) that affect the approach to nucleation, the nucleation event, and the crystal growth phase (figure 2) for each protein studied. Subsequent to the initial set up, which is performed locally, the crystallization experiment can be manipulated and controlled remotely, and followed by both groups on line. As crystal growth rate experiments for each protein are completed using the Xtal Controller, the resulting protein crystals are analyzed via x-ray diffraction and atomic force microscopy (AFM). The x-ray analysis is conducted at the Deutsches Elektronen-Synchrotron (DESY) in Hamburg, Germany using the high brilliant radiation of the storage ring PETRA III, and at the Argonne Synchrotron Facility in Chicago, Illinois. All AFM studies are conducted by Drs. DeLucas and Martyshkin using the facility available in Dr. Martyshkin’s lab in the Physics Building at UAB. A portion of crystals produced in Dr. Betzel’s laboratory are transported to the DeLucas’ laboratory in special thermally insulated containers by Dr. Betzel. In the event that freshly grown crystals must be analyzed prior to one of the planned regular research exchange visits, crystals are to be express mailed to DeLucas’ laboratory in special thermally insulated containers (a routinely used method to transport crystals maintained at 4°C or 22°C without any problems).

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Applications

Space Applications
Researchers have crystallized thousands of proteins, but many of them are not high enough quality to allow scientists to view the proteins three-dimensional structure. One class of proteins, membrane proteins, represent are potentially valuable targets for development sources of new drugs to treat disease, and previous research has suggested that microgravity may improve the quality of this class of important proteins. demonstrated they form high-quality crystals in microgravity. This investigation improves understanding of the physical processes that enable high-quality crystals to grow in space, where Earth’s gravity does not interfere with their formation.

Earth Applications
Crystallizing proteins allows scientists to determine their three-dimensional structure, which enables a better understanding of how proteins work and how they are involved in disease. Protein structure can be used to design new drugs that interact with the protein in specific ways. This investigation provides new insight into how microgravity affects protein crystal growth and quality, benefiting researchers studying protein structure to create new drugs to fight diseases.

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Operations

Operational Requirements and Protocols

The capillaries are maintained at -80°C (using a POLAR -80°C freezer) until activation. The activation process involves removing the capillaries from the freezer and allowing them to thaw out at the ISS ambient temperature. Once activated, the crystals are maintained on an x-y-z translation stage within the LMM. Microscopic observation using the bright field microscope is performed periodically (interval time TBD) over a period not less than 3 days, and not more than 14 days). Approximately 3 to 5 observations per 24 hour period is performed (each observation involves photographic still images, and if possible downlink of the images). For each capillary, it would be helpful to image all of the growing crystals at low and high magnifications. To expedite the time required to observe specific crystals, it is desirable to have the LMM microscope (or alternatively, the x-y-z translation stage) to incorporate the capability of automatically locating previous crystals within each capillary. The translation stage must enable back lighting to optimize crystal viewing. At the completion of observation and microscopic imaging in the LMM, the cassette containing the crystals should be maintained at ~22°C +/- 2.0°C (using a MERLIN incubator, or if not available, using the ambient temperature of the ISS.

The following procedural sequence is to be followed to perform the protein crystallization experiments:
 
Experiment 1: Investigation of Protein Crystal Growth Rates
  1. A cassette containing 5 to 20 frozen capillaries housed in an observation cassette is removed from a -80°C freezer. There are a suite of General Laboratory Active Cryogenic ISS Experiment Refrigerators (GLACIER) - requires two middeck lockers of space) and Polar (requires one middeck locker space) freezers maintained on the ISS.
  2. The capillary cassette is allowed to thaw at ambient ISS temperature, and placed in the LMM for crystallization observation (once the frozen solutions containing within the capillaries have thawed the crystallization processes automatically initiated).
  3. The cassette is positioned under a bright field microscope, and images recorded at 6 to 10 hour intervals depending on real-time observation of the crystal growth rate in microgravity for each particular protein. It is desirable for the software that controls the XYZ translation stage containing the capillary cassette to automatically reposition itself to crystals photographed during previous sessions (this is important so that at higher magnifications the growth rate of individual crystals can be photographically documented). It is also desirable for the images to be downlinked to the ground-based science team.
  4. After a period of 7 to 14 days the crystals stop growing (the exact total growth period will depend on the crystal growth rate for each protein and the protein concentration in each protein solution). At this point the cassette can be removed from the LMM, and either left at ambient ISS temperature, or if available placed in an incubator maintained at approximately 22°C.
Experiment 2:  Investigation of the Influence of Protein Aggregates on Crystal Quality
  1. The procedure followed for this set of experiments is identical to that described for Experiment 1, with the exception that the cassette containing the protein solutions are placed under the LMM's fluorescent microscope.
  2. The cassette used for this set of experiments contains proteins that have different fluorescent probes (fluorophores) with differing excitation wavelengths covalently attached to different aggregate populations of the identical protein. As the crystal begins to grow, the investigation monitors the absorption/excitation of the crystals. It is anticipated that the absorption/excitation is affected depending on the percentage of monomer versus dimer versus trimer of protein aggregates incorporated into the growing crystal.
At the conclusion of the mission, the quality of the microgravity-grown crystals is compared to the control crystallization experiments performed in 1g. The comparison includes:
  1. Microscopic images that support evaluation of crystal growth rates performed in each environment
  2. Fluorescent microscopic images that support evaluation of the percentage of aggregate populations incorporated into crystals grown in both environments
  3. X-Ray diffraction analysis comparing the diffraction resolution with diffraction peak widths (mosaicity) for crystals grown in both environments
Note: All experiments (µg and 1g) for each protein are performed using identical batches of purified protein.
 
 

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Decadal Survey Recommendations

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Results/More Information

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Imagery

image Example of Square, optically transparent capillaries.
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image Example of Square, optically transparent capillaries.

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