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Breathing easier, living longer are goals
of Shuttle experiments

Protein crystal growth experiments
continue on STS-95

November 3, 1998: Easier breathing for infants and adults as well as basic research are on the menu as the crew of the STS-95 Space Shuttle mission tend two sets of veteran experiments that use space to get a better look as the basic mechanics of human health.

Soon after launch, the crew aboard Space Shuttle Discovery activated the Protein Crystallization Apparatus for Microgravity (PCAM) and the second-generation Vapor Diffusion Apparatus (VDA-2) so they could start growing - slowly - crystals of several proteins of interest to researchers on Earth. Both devices have flown several times on the Shuttle, often yielding intriguing results.

Right: An earlier PCAM mission allowed scientists to develop this model of mutant HIV protease - which helps the AIDS virus resist drugs - coupled with an inhibitor that blocks its function. Ground-based research was unable to produce crystals that would let scientists deduce the structure. Microgravity research allowed scientists to determine the shape of the combined proteins. Image courtesy of New Century Pharmaceuticals.

Protein Crystal Growth is a significant research field sponsored by NASA's Marshall Space Flight Center to expand research into the structure and nature of protein molecules and other macromolecules.

Proteins are important, complex biological chemicals which serve a variety of functions in all living organisms. Determining their molecular structures will lead to greater understanding of their functions in living organisms. Many proteins can be crystallized and their molecular structures determined through analysis of those crystals by X-ray crystallography. Unfortunately, crystals grown in the 1-g environment of Earth often have internal defects that make such analysis difficult or impossible. As demonstrated on Space Shuttle missions since 1985, some protein crystals grown in space are larger, and have fewer defects than the Earth-grown counterparts.

Among the many proteins aboard STS-95 is the Respiratory Syncytial Virus (RSV) antibody. RSV is a serious, potentially deadly, disease that usually strikes infants and young children. Understanding the structure of the RSV antibody will help scientists develop pharmaceuticals that help the body fight the disease faster and more efficiently. PCAM is carrying several samples that will be analyzed after the mission.

Both children and adults are often afflicted by allergic reactions to various bits of dust, pollen, animal hair, and other natural bodies. The VDA-2 is carrying grass pollen allergen as part of a study of how it induces allergic rhinitis - an inflamed nose - and bronchial asthma. It is a target for drugs to alleviate allergic reactions. PCAM is carrying other allergens for similar work.

Other specimens will address health issues like AIDS, Venezuelan equine encephalitis, and liver regeneration. Other will support work in fundamental biochemistry.

A complete list of the candidate specimens for STS-95 is at the end of this story. Many of the specimens have flown on earlier missions. The nature of the research in protein structure often requires growth of many specimens to determine the best growth conditions, and to provide enough samples for adequate analysis under X-rays and even neutron beams.

Crystallization methods

PCG apparatus aboard STS-95 will grow crystals by two different methods-vapor diffusion and liquid/liquid diffusion-in two different devices: Second-generation Vapor Diffusion Apparatus (VDA-2) and Protein Crystallization Apparatus for Microgravity (PCAM). In either, the protein under study is dissolved in a solution which is changed to produce conditions which cause the proteins to crystallize.

Both use vapor diffusion which is derived from the "hanging drop" method used extensively on Earth and in the NASA PCG program. In vapor diffusion, almost half the water from a protein droplet moves from the droplet to a reservoir solution. As the droplet becomes more concentrated, crystallization starts.

Experiments with the VDA on earlier missions have helped scientists define procedures and mixtures for efficient growth of protein crystals in space. VDA-2 is designed to improve the mixing of precipitant and protein, and thus enhance crystal growth. Using lessons from earlier missions, PCAM will provide a means for growing larger quantities of crystals with less crew monitoring.

Growing protein crystals requires a well-controlled thermal environment. The PCAM and VDA-2 units will be housed in Single-locker Thermal Enclosure Systems (STES). Each STES takes the volume of a single middeck locker-51.6 x 46 x 27.2 cm (20.3 x 18.1 x 10.7 in)-and supports approximately 12.7 kg (28 lbs) of experiment apparatus.

STES has a 4-button keypad with a small data display and a 30-day, 272-kilobyte data logger. Temperature is controlled by forced-air cooling and by thermal-electric units which use the Peltier effect to move heat. (The Peltier effect produces or removes heat by an electric current flowing through the junction of two dissimilar metals, depending on the direction of the current.)

Second-generation Vapor Diffusion Apparatus (VDA-2)

The VDA-2 employs a three-barrel syringe with protein solution in the first barrel and precipitant solution in the second; the third is used as a mixing chamber on orbit. Each syringe protrudes into an windowed experiment chamber surrounded by an absorbent reservoir. Each VDA-2 tray contains 20 experiment chambers, for a total of 80 chambers.

Syringe tips are plugged before experiment activation and after deactivation. After reaching orbit, a crew member uses a torque driver to withdraw the plugs. Then another mechanism is turned to move the pistons inside the first two barrels of each syringe.

Left: A single VDA-2 syringe is little bigger than a penny. The droplet forms at the tip at bottom right. Links to larger image.

This pushes protein and precipitant solutions onto the tip of the syringe, where they combine to form a droplet approximately 40 mL in volume (about 4.2 mm [1/6th inch] in diameter). A piston in the third barrel (unique to the VDA-2 design) pulls the droplet into the empty barrel, then pushes it back out. This is repeated several times to ensure complete mixing. The mixed droplet remains on the syringe tip during the vapor diffusion process.

The vapor pressure difference between each drop and its reservoir solution causes water vapor from each drop to move into the reservoir solution. This method, called vapor diffusion, is the most commonly used method for growing protein crystals. As water is removed from the drop, protein molecules form crystals. Growth stops about one day before landing when the droplets are drawn back into the syringes and the tips are plugged.

VDA-2 was developed by the Center for Macromolecular Crystallography at the University of Alabama in Birmingham under the sponsorship of MSFC.

Protein Crystallization Apparatus for Microgravity

The basic PCAM design places a protein sample drop in a small well surrounded by an absorbent reservoir. A synthetic rubber (elastomer) seal isolates the drop from the reservoir except during crystal growth while in space.

Right: Two PCAM trays would easily fit in the palm of your hand. Links to larger image.

A single, full-length PCAM cylinder is 81 mm (3.2 in) in diameter and 381 mm (15 in) long. Each cylinder contains nine trays held in position by guide rods and separated from each other by springs. The trays are sealed by the elastomer seal which has adhesive to bind it to the upper face of the tray around the wells. A tray, in turn, holds seven sample wells, each surrounded by a donut-shaped reservoir which absorbs the precipitant solution.

Comparison of crystals grown on Earth and in space. Both are in PCAM on STS-95.


1-g


1-g


Microgravity
Augmenter of Liver Regeneration


Microgravity
Neurophysin-Vasopressin Complex

B. C. Wang & J. Rose/biocrystallography/Univ. of Georgia. Images courtesy of New Century Pharmaceuticals

From laboratory preparation until the start of the experiment in space, the elastomer seal is pressed by a metal plate so it isolates each samples from rest of its chamber. To start the experiment, an astronaut opens the front of the STES and, using socket wrench, rotates a shaft on the end of the cylinder clockwise.

This lets the seal retract and allow vapor diffusion to start. When all six cylinders are activated, the astronaut closes the STES. Before the end of the mission, and the astronaut rotates the shafts counterclockwise to reseal the samples before return to Earth. The samples remain sealed until they are returned to the laboratory for analysis.

PCAM trays have flown on several missions to date and have demonstrated that this is an effective means for screening large quantities of proteins for detailed study later. PCAM was developed at the MSFC Laboratory for Structural Biology.

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Web Links

On target for a cure provides more details of the PCG program.

External Links:
New Century Pharmaceuticals- is responsible for research with the PCAM.
Center for Macromolecular Crystallography at the University of Alabama in Birmingham is responsible for research with the VDA-2.

Candidate samples

Protein samples are selected by a committee chaired by the Biotechnology Discipline Scientist, Space Science Laboratory, MSFC. Samples are then evaluated and approved by NASA toxicology and safety offices.

As a point of comparison, the molecular masses of proteins range from about 890 to 2,200 times that of ordinary sugar, a relatively simple organic compound which is easily crystallized.

PCAM candidate samples

Dr. Daniel Carter (Principal Investigator), New Century Pharmaceuticals (NCP)

  • Respiratory Syncytial Virus Antibody, Therapy and vaccine development
  • Lysozyme, Crystal growth model system
  • Albumin, Drug delivery

Dr. Jean-Paul Declercq, University of Louvain, Belgium

  • Parvalbumin, Fund. biochemistry

Dr. John Rosenberg, University of Pittsburgh

  • E.coli Gro EL, Fund. virus structure & function
  • Eco RI Endonuclease-DNA complex, Fund. biochemistry
  • Mycobacterium L5 gp71 Repressor, Fund. biochemistry
  • CFA1 Pilin, Biochemistry; therapeutic development

Dr. Dennis Bamford, University of Helsinki, Finland

  • Bacteriophage PRD1, Biotechnology & biochemistry

Dr. Bill Thomas, University of Alabama in Huntsville; Dr. Daniel Carter, NCP

  • Ferritin/Apoferritin, Fund. biochemistry; crystal growth model system

Dr. B.C. Wang, Dr. John RoseUniversity of Georgia

  • Augmenter of Liver Regeneration, Accelerates liver cell regeneration
  • T7 RNA Polymerase, Fund. biochemistry
  • Neurophysin II/Vasopressin Complex, Fund. biochemistry

Dr. Rhoda Hirsch, Albert Einstein College of Medicine; Dr. Bill Thomas, University of Alabama in Huntsville; Dr. Daniel Carter, NCP

  • Hemoglobin C, Fund. biochemistry, sickle cell anemia

Dr. Wolfgang Weber, University Eppendorf, Germany

  • Pollen allergen, Fund. biochemistry, therapeutic development

VDA-2 candidate samples

Dr. Christian Betzel, University of Hamburg, Hamburg, Germany

  • Proteinase K/substrate Complex, Structural data for designing inhibitors for pharmaceutical applications

Dr. Lisa Edberg, Center for Macromolecular Crystallography, Birmingham, Alabama

  • VEE Capsid Protein, Key in the assembly and propagation of Venezuelan Equine Encephalitis virus

Dr. Mark Jedrzejas, Center for Macromolecular Crystallography, Birmingham, Alabama

  • Germination Protease, Germination of organisms that form spores

Dr. Russell Judge, University of Alabama in Huntsville/NASA Marshall Space Flight Center

  • Glucose Isomerase, Converts glucose to fructose; industrial applications

Dr. Edward Meehan, University of Alabama in Huntsville

  • Dehydratase, Applications for immune-compromised patients such as those with AIDS and cystic fibrosis

Dr. Alfonso Mondrag贸n, Northwestern University, Evanston, Illinois

  • RNA Component of RNAse P, Design of drugs that mitigate RNA catalysis

Dr. Stephen Quirk, Georgia Institute of Technology, Atlanta, Georgia

  • 8-oxidodGTPase, Prevents oxidative damage prior to DNA replication

Dr. Wolfgang Weber, University of Hamburg, Hamburg, Germany

  • Grass Pollen Allergen Phl p 5b, Induces allergic rhinitis and bronchial asthma; target for drugs to alleviate allergic reactions

These experiments are managed by the Marshall Microgravity Research Program Office, Biotechnology Project Office. The Biotechnology Discipline Manager is Ron Porter.

Principal investigator for PCAM is Dr. Dan Carter of New Century Pharmaceuticals in Huntsville, Ala. The project manager is Keith Higginbotham of the MSFC Biotechnology Project Office.

Principal investigator for the VDA-2 investigations is Dr. Larry Delucas of the University of Alabama in Birmingham. The Project Manager is Dewayne Collins of the Biotechnology Project Office, MSFC.

More web links

More Space Science Headlines - NASA research on the web

Microgravity Program Office at Marshall Space Flight Center

Life and Microgravity programmatic information from NASA headquarters.


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Author: Dave Dooling
Curator: Bryan Walls
NASA Official: John M. Horack