November 17, 2005
Zooming in on the Protein-Conducting Channel
Researchers have gained the most detailed view yet of the heart of
the translocon, a channel through which newly constructed proteins are
inserted into the cell membrane. The process of transporting proteins
across or into membranes is a critical function that occurs in every
cell.
Howard Hughes Medical Institute investigator Joachim Frank at the
Wadsworth Center and his colleagues reported their detailed study of
the translocon's core, called the protein-conducting channel (PCC), in
an article published in the November 17, 2005, issue of the journal
Nature. Co-lead authors on the paper were Kakoli Mitra in
Frank's laboratory and Christiane Schaffitzel of the
Eidgenössische Technische Hochschule Hönggerberg in
Switzerland, who is in the laboratory of the other senior author, Nenad
Ban. Other co-authors were from the Scripps Research Institute and the
State University of New York at Albany.
“What we have achieved is a huge jump in resolution of this complex.”
Joachim Frank
The researchers studied the PCC, which grabs newly made protein as
it is extruded from the ribosome's protein synthesis machinery. The PCC
then opens either a pore that is perpendicular or lateral to the cell
membrane to feed the new protein either across or into the
membrane.
For the studies, the Swiss researchers created a complex comprising
the PCC from E. coli attached to a ribosome that
contained a newly forming protein segment. The ribosome is the massive
protein-RNA complex that constitutes the cell's protein-making
machinery.
Mitra explored the structure of this PCC-ribosome complex using
three-dimensional cryogenic electron microscopy (cryo-EM), as well as
computational methods. Three-dimensional cryo-EM is one of the few
techniques capable of visualizing large, dynamic molecules.
In preparing for cryo-EM, researchers immersed the PCC-containing
complex in water and then abruptly froze it in supercold liquid ethane.
The rapid freezing imprisoned the complex in vitreous ice, a glassy
non-crystalline form of ice, thus preserving its native structure.
Using an electron microscope with a low-intensity beam to avoid
damaging the molecules, scientists then obtained images of thousands of
captive protein complexes. Next, they used computer image analysis to
produce detailed, three-dimensional maps of the complex in two
different states from the low-contrast, noisy images produced by the
electron microscope.
“What we have achieved is a huge jump in resolution of this
complex,” said Frank. “Even so, this resolution would not
allow us to study the complex in atomic detail, or even see individual
helices.” He said the results from the cryo-EM analysis were
informed by detailed x-ray crystallographic data on the PCC structure
done by other researchers. In x-ray crystallography, an x-ray beam is
directed through crystals of a target protein. As the x-rays pass
through the crystal, they are diffracted. Researchers can then analyze
the diffraction pattern to determine the atomic structure of the
protein.
The analysis by Frank and his colleagues revealed that each channel
consists of two PCC subunits joined in a clamshell arrangement. The
cryo-EM data also revealed two different arrangements of the PCC — one
that was apparently in the functional, or “translocating”
state, and one in a non-translocating state.
X-ray crystallography data from the lab of HHMI investigator Tom A.
Rappaport suggested that the halves of the PCC clamshell were joined in
a back-to-back arrangement. However, said Frank, x-ray crystallographic
structures often do not represent the arrangements of proteins in their
native functional state.
Thus, he and his colleagues applied a computational analytical
method called “normal mode-based flexible fitting” (NMFF)
to model how well the two possible channel structures could explain the
structural data from cryo-EM. The NMFF method was developed and applied
by co-authors Florence Tama and Charles Brooks of the Scripps Research
Institute. The technique provides dynamic information on the multitude
of vibrations and motions that complex molecules preferentially
undergo.
NMFF analysis revealed that the cryo-EM data were best explained by
a model in which the two PCC clamshells were joined in a
“front-to-front” arrangement. This arrangement yielded
significant insight into how the channel functions to translocate
proteins across or into membranes, said Frank.
“Now that we have these new insights into the architecture of
the PCC in its translocating, and possibly non-translocating state, we
can explore the mechanisms of perpendicular versus lateral
transport,” Frank said.
|
Jennifer Michalowski
