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New technique helps researchers determine amino-acid charge
Jim Barlow, Life Sciences Editor
217-333-5802; jebarlow@illinois.edu
12/16/2005
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CHAMPAIGN, Ill.
— Measurements
of the ion-current through the open state of a membrane-protein’s
ion channel have allowed scientists at the University of Illinois at
Urbana-Champaign to obtain a detailed picture of the effect of the protein
microenvironment on the affinity of ionizable amino-acid residues for
protons.
The findings, reported in the Dec. 15 issue of Nature, are expected
to be welcome news for chemists and biophysicists, both experimentalists
and theoreticians, because they have previously relied on theoretical
estimations to predict protonation states – whether an amino acid
is charged or not. Appropriate experimental benchmarks also had been
lacking.
All cells have membrane proteins that form channels to allow water and/or
ions to pass through. Malfunctions have been linked to such problems
as hypertension, abnormal insulin secretion, abnormal heart conditions
and brain seizures. As a result, these membrane proteins are often targeted
by drug treatments.
Previous approaches didn’t provide sufficient resolution to let
researchers accurately detect the association and dissociation of protons
to and from individual amino-acid residues in real time.
Using the patch-clamp technique, the researchers were able to probe
the electrostatic properties of the inner lining of the ion-channel’s
pore, and, from there, they inferred the rotational angle of the pore-lining
alpha-helices in the open state.
In this case, researchers focused on the muscle nicotinic acetylcholine
receptor, a membrane protein that mediates voluntary muscle contraction.
“Our paper has implications that are specific to this receptor,
but many of the findings can be extended to several other membrane proteins,”
said Claudio Grosman, a professor of molecular
and integrative physiology at Illinois.
“We are working with the open state of the ion channel, and we
now know how the helices that line the pore are oriented. This was not
known before,” Grosman said. “Previous work has told us
how the helices are oriented in the closed state.”
A major problem in understanding the relationship between structure
and function in proteins, and the impact that electrostatics have on
them, Grosman said, is not knowing the protonation state of ionizable
residues. Protons, he added, are so small that they cannot be detected
even with X-ray crystallographic approaches.
For the study, Grosman and colleagues used protein engineering. They
mutated each residue of the pore’s lining with basic amino-acid
residues, which can acquire a positive charge upon binding protons and
become neutral upon releasing them.
“One thing is the proton affinity of an amino-acid residue when
the amino acid is dissolved in a lot of water in, say, a glass beaker;
another thing is the affinity for protons in the complex microenvironment
presented by a membrane protein,” Grosman said. “For the
first time, we were able to measure proton-transfer events at the single-proton,
single-amino-acid level, in real time. Chemists will be happy to see
this.”
In addition to providing an extensive set of proton-affinity values
for basic residues, which differed greatly from those found in a “glass
beaker,” the findings also discounted a long-held theory that
the rotation of the pore-lining helices underlie the mechanism of opening
and closing of the nicotinic receptor ion channel. The data, Grosman
said, indicate that such rotation is minimal, if any.
Co-authors of the study were Gisela D. Cymes, a postdoctoral researcher,
and Ying Ni, a research specialist, both in the department of molecular
and integrative physiology. The three researchers also are part of the
Center for Biophysics and Computational Biology and the Neuroscience
Program at Illinois.
The National Institutes of Health funded the research.
