Blasting Antibodies with Lasers Provides Direct Way of Measuring
By Jason Socrates
A group of scientists at The Scripps Research Institute
(TSRI) and San Diego Supercomputer Center at the University
of California at San Diego (UCSD) have used a powerful laser
in combination with innovative quantum mechanical computations
to measure the flexibility of mouse antibodies.
The new technique, described in an upcoming issue of the
journal Proceedings of the National Academy of Sciences,
is significant because protein flexibility is believed to
play an important role in antibody-antigen recognition, one
of the fundamental events in the human immune system.
"This is the first time anybody has ever gone into a protein
and experimentally measured the frequency of protein vibrations
in response to an applied force," says Floyd Romesberg, assistant
professor in the Department of Chemistry at TSRI, who led
"Our results show that the motions of the antibody-antigen
complexes can range over four orders of magnitude, from tens
of femtoseconds to hundreds of picoseconds," says co-author
Kim K. Baldridge, director of Integrative Computational Sciences
at the San Diego Supercomputer Center and an adjunct professor
of Chemistry at UCSD. "This is evidence of a general mechanism
of antigen-antibody interactionswhich range from rigid
to flexible," she adds.
Flexibility of Proteins
Protein flexibility is an important concept in biology because
of its role in protein-protein and protein-ligand recognition.
One of the longest running debates in molecular recognition
is how proteins recognize and bind to other moleculeswhether
it resembles putting a key into a lock (the lock and key model)
or catching a baseball in a catcher's mitt (the induced fit
There are lots of ideas about mechanisms of antigen recognition
postulated in the literature, but what the debate comes down
to is really a question of flexibility. How flexible are proteins?
Unfortunately, flexibility is difficult to characterize
experimentally, and there has never been a study like this
one to carefully examine the details of antibody recognition
of antigen at the molecular level, which involves bond vibrations
that ever-so-slightly displace atoms a million times every
millionth of a second. Scientists have had a tough time studying
these vibrations because the two main techniques that allow
them to "look" at proteinsnuclear magnetic resonance
(NMR) and X-ray crystallographycannot be used.
X-ray crystallography provides only average structures,
which provide no direct information on a protein's flexibility.
NMR can, in principle, be used to measure a protein's flexibility,
but in practice is limited to slow timescales, involving large
amplitude motions. Moreover, the number of atoms within a
molecule the size of an antibody is so large that drawing
conclusions from the data is nearly impossible.
Now Romesberg, Baldridge, and their colleagues have developed
a way to measure the flexibility of proteins over timescales
ranging from femtoseconds to nanoseconds using a combination
of spectroscopy and quantum mechanical techniques.
Shaken Not Stirred
For years biochemists have routinely used bench top ultraviolet
and visible light spectrometers to measure things like protein
concentration or to follow chemical reactions. However, Romesberg's
spectrometer is not the kind you might find in any catalog
of equipment lying around the lab.
This laser, built by Romesberg and Research Associate Ralph
Jimenez, Ph.D., takes up an entire room and emits a burst
of photons in a roughly 50-femtosecond pulsewhich is
billions of times faster than the fastest shutter speed on
a good camera. This incredible speed is necessary, because
just as a fast shutter speed captures a fast movement on film,
a fast laser captures a fast movement within a protein.
"The laser allows us to take 'photographs' of a protein
vibrating," says Romesberg.
In their experiments, Romesberg and Jimenez mix human antibodies
with dye molecules. When the mixture is blasted with the laser
beam, the dye molecules absorb energy from the laser and transmit
some of this energy into the antibody.
The only place for the energy to go within the antibody
is for it to be absorbed by vibrating bonds within the protein.
The electron distribution in these bonds may then change,
depending on how much they vibrate. By comparing an excited,
"spectra" readout to a normal spectrum, Romesberg and his
colleagues can assess how flexible particular parts of a protein
This is not always simple. Antibodies are large proteins
with lots of vibrating bonds, and molecular motions. Quantum
mechanical calculations can help researchers delineate which
motions are primary participants in the antibody-antigen recognition
process. Baldridge took the results of these computations
and provided a visual way to understand the effect of the
force on the protein.
The quantum mechanical calculations actually give a depiction
of the electrostatic processes that are occurring. Together
with the experimental information, this helps complete the
puzzle of how various bonds are moving, twisting, and interacting
with other atoms in the protein environment.
The lock-and-key model specifies that if the antigen and
antibody are not matched up in a rigid, structural way, they
will not bind. Romesberg, Baldridge, and their colleagues
found this to be true for one of the antibodies they tested.
But two of the other antibodies appeared to wiggle a lot to
achieve their optimal energetic state.
Antibody recognition, says Romesberg, may not be a simple,
lock-and-key mechanism, but one in which the keys and the
locks are vibrating and changing their shape as they come
together in solution.
The article, "Flexibility and Molecular Recognition in the
Immune System" was authored by Ralph Jimenez, Georgina Salazar,
Kim K. Baldridge, and Floyd E. Romesberg, and appears in the
online edition of the journal Proceedings of the National
Academy of Sciences the week of December 16, 2002. The
article will appear in print in early 2003.
This work was supported by The Skaggs Institute for Chemical
Biology, the National Science Foundation, and the National
Institutes of Health.