beads make sensitive assay for protein drug candidates
Robert Sanders, UC Berkeley Media Relations
glass beads wearing coats identical to the outer membrane of a cell
provide a powerful assay for proteins that bind to cell membranes,
such as protein drugs or drug candidates, according to chemists
at the University of California, Berkeley, and Lawrence Berkeley
National Laboratory (LBNL).
The membrane-coated beads, complete with receptors that dot the
surfaces of real cells, also would make a sensitive detection system
for viruses or protein toxins like those produced by cholera, anthrax
and tetanus bacteria.
detection system is extremely sensitive - when proteins latch onto
receptors in the membrane, the coated beads disperse like pool balls
after a break. This dispersal can easily be seen through a microscope,
making robotic screening possible.
a big demand for membrane-based detection systems by pharmaceutical
companies and researchers, but a final bottleneck is always getting
a biochemical measurement from the membrane change - detecting the
change," said Jay T. Groves, professor of chemistry
at UC Berkeley and a faculty scientist in the Physical Bioscience
Division of LBNL. "The hard thing about detecting molecules
on surfaces is that a surface is intrinsically two-dimensional,
and there simply are not many molecules there to detect. For this
reason, sophisticated techniques have generally been required to
analyze molecular binding events on membrane surfaces.
our technique, you get the best of both worlds. We have single molecule
interactions that you can see without a sophisticated instrument."
beads essentially amplify the tiny effects caused by binding of
a protein to a receptor, he said. Groves and biophysics graduate
student Michael M. Baksh explain their detection scheme in
a paper appearing in the Jan. 8 issue of Nature.
is a member of the California Institute for Quantitative Biomedical
Research (QB3), a cooperative effort among UC Berkeley, UC San Francisco
and UC Santa Cruz to leverage strengths in the physical and biological
sciences and engineering to improve human health and the environment.
a detection system or assay would be of great interest to the pharmaceutical
industry, which needs to screen drug candidates for how well they
bind to specific membrane-bound receptors, Groves said. That's because
many drugs as well as infectious disease organisms target memranes.
companies typically screen hundreds of thousands of compounds -
and occasionally a million of them - at a time, an automated and
easy-to-read assay would save time and money.
is a very high-throughput process that pharmaceutical companies
could easily incorporate into their robotic systems for high-throughput
screens," Groves said.
has been working on ways to simplify the study of cell membranes,
and several years ago came up with a "MembraneChip" -
a piece of cell membrane, complete with receptors, attached to a
silicon electronic chip that could read out a change in the membrane
caused by the binding of a protein with its receptor. Groves patented
the device and founded a company, Synamem Corp., that licensed the
original technology to look for new drugs that suppress the body's
immune response or fight infection.
new technique is a MembraneChip on a particle, and it will probably
replace the MembraneChip in many applications because it is very,
very high throughput," he said.
membranes consist of two layers of fatty molecules called lipids.
The lipid bi-layer protects the cell contents but, just as importantly,
provides a sea in which big molecules - receptors - float as entry
portals for specific proteins. What Groves and many others have
tried to do is find an easy way to determine how well a given protein
binds to a membrane-bound receptor. Drug companies, for example,
want chemicals that fit like a hand in a glove, to either stimulate
or block a receptor.
is tremendous interest in measurement and analysis of molecular
binding events that occur on cell membrane surfaces," he said.
"A substantial majority of therapeutic drug targets reside
in the membrane, as do recognition targets used by viruses to infect.
present, though, it is very difficult to measure binding interactions
at membrane surfaces. Elaborate techniques such as surface plasmon
resonance (SPR) or total internal reflection fluorescence (TIRF)
must be employed."
reading about recent research on colloids - microscopic particles
that cluster in interesting ways - Groves thought that the degree
of aggregation of colloids might make a good indicator of receptor
binding affinity, if the colloidal particles were coated with a
membrane and receptors.
experiments showed that a colloidal suspension of membrane-coated
beads does react to the binding of a protein and receptor. When
no proteins are present, the beads wander randomly through a thin,
flat layer of fluid, clustering in short-lived groups of a dozen
membranes make the beads slippery, so they don't stick together
but slide around one another," Groves said. "The clumps
are just random clusters."
addition of a protein that fits into a receptor, bead movement is
disrupted so that the random clusters shrink in size. The response
is not all or nothing, but is more pronounced the more tightly the
protein fits into the receptor.
proteins that do not fit into a receptor do not elicit this reaction,
the system provides a sensitive screening test for presence of specific
colloid is poised near a phase transition, making it very sensitive
to single particle interactions too small to measure individually,"
he said. "The population behavior of the colloid gives us subtle
information about membrane interactions not accessible in other
beads are off-the-shelf glass (silica) spheres 5 microns across,
about 1/20th the width of a human hair. The membrane is only 5 nanometers
thick, 1,000 times smaller than the diameter of the bead.
practice, a robotic scanner would need to look at a window only
a few millimeters square and calculate the pair-wise distances between
the centers of all beads. The shape of this distribution tells how
much the beads are aggregating, and thus indicates the binding affinity
of a protein to a receptor. Groves himself plans to use the membrane-coated
beads to study the dynamics of receptors in cell membranes.
science of this technique is intriguing," writes Thomas M.
Bayerl in a commentary appearing in the same issue of Nature. Bayerl
is at the Physical Institute at the University of Würzburg,
Germany. "This work by (the UC Berkeley/LBNL team) may open
the door to the automated characterization of a wide range of complex
molecular interactions that are at present poorly understood."
third author of the paper is Michal Jaros, a visiting Fulbright
graduate student fellow at UC Berkeley who has since returned to
Charles University in Prague in the Czech Republic.
work was supported by the U.S. Department of Energy and by a Burroughs
Wellcome Career Award in the Biomedical Sciences to Groves.