SNS and biological research
Some drugs that combat AIDS—the 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 infections—have 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
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
"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.