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Proteins' Subtle 'Backrub' Motion Could Have Important Implications

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Duke Health News 919-660-1306

DURHAM, N.C. -- Biochemists have detected a surprising,
subtle new gyration that protein molecules undergo in the
intricate, squirming dance that influences their activity in
the cell. The researchers have also created a realistic
geometrical model of the twisting "backrub" motion that could
help scientists understand the basics of protein function and
design proteins for medical use.

Also, they said, the backrub motion could have implications
for understanding how proteins can accommodate locally to some
mutations that occur during evolution, without altering their
global structure or function.

Understanding the subtleties of protein motion is important
because the molecules are central to the machinery of life. For
example, protein enzymes catalyze the myriad of chemical
reactions that underlie all cell functions. Thus, biologists
seek not only to understand the complexities of protein
dynamics, but to design and construct manmade proteins as
medicines to treat a wide array of diseases.

The Duke University Medical Center biochemists, led by
Professors Jane and David Richardson, published their findings
in the February 2006 issue of the journal Structure. Lead
author on the paper was graduate student Ian Davis, and the
other co-author was Bryan Arendall. The research was supported
by the National Institutes of Health and a Howard Hughes
Medical Institute predoctoral fellowship to Davis.

Proteins comprise strings of amino acids whose links form a
"backbone." Each kind of amino acid sprouts a characteristic
molecular "side chain," and together the backbone and side
chains determine a protein's structure and function.

The Duke researchers suspected the presence of backrub
motions for other reasons, but their reality could be
conclusively shown only by studying proteins frozen in
crystalline form for structural analysis by x-ray
crystallography. In this widely used technique, x-rays are
directed through crystals of a protein, and the pattern of
diffracted beams is analyzed to deduce its structure. Such data
is usually collected at synchrotron x-ray sources, with the
crystals cooled by liquid nitrogen to temperatures near -300°
Fahrenheit.

"We were pleased but surprised that these crystal structures
at liquid nitrogen temperatures actually could show us
something really interesting about dynamics," said Jane
Richardson.

In the highest resolution such crystal structures, in which
individual atoms are directly visible, it is quite common to
see a side chain that "dances", or flips back and forth between
two different conformations. The researchers traced the
consequences of this motion back into the backbone and deduced
that the local backbone structure must twist slightly in a
particular way to accommodate the larger side-chain movement.
According to Richardson, this motion, which they dubbed a
"backrub", is a subtle, concerted shift of the two backbone
units on either side of the dancing side chain.

"Nobody has described this particular kind of motion
before," said Richardson. "And that's because it's down in the
noise, in terms of what the backbone is doing. You have to get
ultra-high-resolution, clean maps that really show you exactly
where the side chain atoms are. And then you can work backwards
to figure out what the backbone must have done." The
researchers created a geometrical "backrub" software tool to
model this motion.

"Investigators had theorized that the backbone moved, but it
has been rather difficult to prove what's really going on,"
said Richardson. "There have been other previous models, but
these were not as successful as one would like, presumably
because they were not based on the kind of empirical data that
we've now developed." To understand how frequently the backrub
motion occurred in proteins, Davis undertook an analysis of
crystallographic data on 19 proteins.

"We took nineteen of the highest-resolution structures
available -- the best data we could get our hands on," said
Davis. "We then went through each of those structures one
residue at a time, looking for evidence of some sort of motion
that extended beyond just a side chain spinning."

Davis distinguished backrub motions by detecting movement of
the first side chain atom attached to the backbone -- a shift
that could only occur if the backbone had moved, said Davis. In
analyzing some 4,000 amino acid units, Davis found backrub
motion for more than 3 percent of the total and 75 percent of
all the local backbone shifts.

"We were expecting to see some examples of this motion, but
we were very surprised that it was so dominant over any other
very local backbone motions," he said.

The fact that the backbone motions are common in proteins
frozen in crystals suggests that they are even more prevalent
in proteins in the liquid environment of the cell, said
Richardson.

"The backrub motions in these frozen crystals have to be a
subset of what goes on in solution or in the body," she said.
"It's got to be more common when you have more flexibility."
Such ubiquity means that the backrub model developed by the
researchers could have useful applications, said
Richardson.

"The backrub model can be used by people doing homology
modeling, in which they are starting with a known protein
structure and trying to model the structure of a protein with a
related but different sequence," she said. "One knows that the
backbone will shift in such cases, but previous methods of
modeling those shifts have usually produced results farther
from reality rather than closer.

"We think our backrub shifts can help predict how, either in
natural evolution or in protein engineering, the local
structure would accommodate the substitution of an amino acid
with a different shape or size of side chain," said Richardson.
Such engineering is commonly done in developing altered
proteins for medical use. The Richardsons and their colleagues
are already working with fellow biochemists who design
proteins, to explore how their backrub model can improve design
strategies.

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