Some drugs that combat AIDSthe deadly disease caused by the human immunodeficiency virus (HIV) that kills or damages the body's immune system cells so the body can't fight infectionshave been discovered by accident. For example, the protease inhibitors used to treat AIDS were originally tested as medications for reducing blood pressure. These drugs resemble pieces of the protein chain that the HIV enzyme, called protease, normally cuts. By gumming up the protease scissors, HIV protease inhibitors prevent the protease from cutting long polyprotein chains into the shorter structural proteins HIV needs to assemble a cocoon of protection around its RNA genome. Although the virus can still invade other cells, without the protective cocoon, the virus genome is exposed to host enzymes, which easily destroy the invading viral RNA and prevent further replication.
Finding the right chemical structures to halt the replication of HIV and then bringing the drugs to market can take years and cost millions of dollars. As a result, millions of people who need these medications to survive cannot afford them. However, there is cause for hope. Recent work in biological neutron crystallography and new biological instruments planned for the Spallation Neutron Source (SNS) at ORNL could provide rapid insights into macromolecular structures that could lead to safer, more effective, and less expensive drugs.
Determination of protein structures by neutron diffraction will be useful for drug design. HIV protease, a target of AIDS drug designers, belongs to the general class of protein-digesting enzymes called aspartic proteases. These enzymes (including the stomach's pepsin) are named for aspartic acid, which is present at their active sites. A solvent molecule bound tightly to aspartate carboxyl groups is presumed to take part in the catalytic mechanism that enables the enzyme to break a protein chain. The best currently accepted mechanisms are largely based on X-ray images of inhibitor structures, but the active-site hydrogen atoms cannot be definitively located by current X-ray analyses, leaving an incomplete picture of enzyme catalysis.
Jonathan Cooper of the University of Southampton in England and Dean Myles of the European Molecular Biology Laboratory Outstation in Grenoble, France, published a report in 2000 on the neutron diffraction structure of the fungal aspartic protease endothiapepsin. This work represents the largest protein solved by neutron diffraction methods to date. The success of neutron diffraction in determining the positions of catalytic hydrogens reveals a route to the development of more effective inhibitors to aspartic proteases. It also suggests that this method could play a significant role in rational drug design.
Similar and better atomic-resolution images of biological substances are anticipated from the SNS after its completion in 2006 when it is operating at 2 megawatts. That's because the SNS will have a much higher neutron flux; it will offer 10 times the number of neutrons now available at any existing neutron research facility. The intrinsic time structure of the pulsed neutron source makes SNS ideal for atomic-resolution protein crystallography studies.
"If the SNS has an instrument that can do high-resolution crystallography on protein crystals with 100-angstrom repeats," says Gerard Bunick of ORNL's Life Sciences Division (LSD), "we could contribute crucial molecular information that could lead to more effective drugs against HIV, for example.
"X rays give excellent high-resolution images of heavy atoms in protein crystals, but neutrons also see lighter atoms such as hydrogen, which makes up half of all the atoms in proteins. Only one in 100 proteins crystallizes well enough to get the resolution needed to see hydrogen atoms using the bright X rays from synchrotrons, according to a survey of structures in the Protein Data Bank."
Only about a dozen protein structures have been solved using neutron diffraction. But that could change with the opening in 2001 of the new Protein Crystal Station at the Los Alamos Neutron Scattering Center and the later operation of the SNS. Bunick's colleague Leif Hanson from the University of Tennessee says that within 10 years, 50 to 100 protein structures will be determined annually using neutron diffraction. Neutron diffraction studies of protein structures are also being facilitated by the availability of improved neutron detectors that speed up data collection and the ability to grow larger crystals of proteins.
"It is now easier to grow larger protein crystals in space and on earth," says Bunick, who set up crystal-growing experiments that were run on the U.S. Space Shuttle Columbia in 1995 and on the Russian Mir space station in the late 1990s. Nearly perfect crystals of nucleosomes were grown in the microgravity environment of these vehicles orbiting around the earth. Nucleosomes are the building blocks of chromosomes; they each consist of a core of histone proteins around which approximately two turns of double-stranded DNA are wrapped.
"We have proposed to NASA to grow large protein crystals for neutron studies in the microgravity environment of the International Space Station," Bunick says. "And better ways to grow large protein crystals on earth have been developed as a result of microgravity crystal growth research sponsored by NASA."
Bunick, Hanson, and Joel Harp, all of LSD; Chris Dealwis of the University of Tennessee; and Jinkui Zhao of the SNS organized and co-hosted an SNS workshop December 18, 2000, in Knoxville. They were pleased that the workshop participants recommended that two instruments be designed to do high-resolution protein crystallography studies using SNS neutrons. The first instrument would be used for high-resolution protein crystallography on crystals with up to 100-angstrom (Å) repeating motifs. The second instrument, if funded, would be located in the long-wavelength target station. It would be used to study protein complexes with 200-250 Å repeating motifs in the crystals, other large macromolecular complexes at lower resolution, and biological membrane systems.
According to Thom Mason, ORNL's associate laboratory director for the SNS, biologists are interacting with engineers to design 3 of the 12 world-class scientific instruments planned for the SNS. These instruments will complement the capabilities of DOE's Center for Structural and Molecular Biology at ORNL. They will help biologists determine the atomic-level structure of proteins, amino acids, hormones, peptides, and other signaling compounds that allow cells to communicate with each other and coordinate their activities across the organism.
Besides the protein crystallography instrument, biologists plan to use a liquids reflectometer to study changes in surfaces, interfaces, and layered structures in biological materials. "It could help scientists study how proteins affect the structure of membranes in cells," Mason says.
A small-angle neutron scattering (SANS) instrument is also planned. It will "see" a target with a length scale ranging from thousands of angstroms to less than an angstrom. "It's a little like having a camera with a zoom lens," Mason says. "You zoom in on the subject to see the small details. Then you pull the zoom lens back to get the big picture, but you can't see the smallest details. With the SANS at the SNS, we will be able to zoom in on the protein's details at the interatomic level, what we call large Q. Then we can pull back and see the whole protein, what we call small Q. We can span this wide range in a single experiment, something that can't be done easily with X-ray crystallography or existing neutron instruments."
Zhao, a neutron scientist who does biological studies at the High Flux Isotope Reactor (HFIR), says that the biological SANS instruments that will be installed at both HFIR and the SNS will be used to study proteins and other biological molecules in their natural solution. Both instruments will be superbly suited to study the shape, conformational changes, and dynamics of proteins. "The difference will be that the HFIR SANS will look at large length scales, whereas the SNS SANS will look at a range of length scales simultaneously, such as protein-membrane interactions, with proteins on the large-length-scale side and membranes on the small-scale side."
"To study the interactions of proteins and other biological molecules in solution, we must use contrast matching," Zhao says. In this technique, some hydrogen atoms in the sample are replaced with heavier hydrogen, or deuterium, atoms. Deuterium and hydrogen atoms scatter neutrons very differently. Changing the ratios of hydrogen and deuterium in the solvent water mixture changes the visibility of proteins to neutrons. It's like adding red dye to a glass of water containing red and yellow balls so that all you see are the yellow balls."
"Scientists mask out the portion of the protein they are not interested in and make highly visible the active part of the protein that does interest them," says Mason. "The ratios of hydrogen and deuterium in water mixtures can be changed in various samples to meet researcher needs."
The SNS can also be used for inelastic neutron-scattering experiments for the study of protein dynamics. Inelastic scattering of neutrons results from an "inelastic collision" in which the total kinetic energy of neutrons colliding with target atoms is not the same after the collision as before.
"Measurements of neutron-scattering energies will provide information on the collective motion of proteins, which can be correlated with protein function," Zhao says. "For example, when a catalytic protein is active, its motion is random. But when its motion becomes harmonic at low temperature, its catalytic function stops."
Bunick and his colleagues could use an SNS protein crystallography instrument to help them better understand the origin of Rett Syndrome (RS), a debilitating neurodevelopmental disorder that causes loss of speech and profound mental retardation in young girls. Studies at the SNS might improve understanding of the structure and interactions of native and mutant DNA-binding proteins, enabling the production of drugs to reverse the deleterious effects of the defective protein.
When research comes to life at the SNS, we can expect some early discoveries to jump-start the design of drugs to improve human life.
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