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Proteins, the building bocks
of the organism, generally function through mutual cooperation. They bind
together into protein complexes and help each other to fulfill their functions.
Although researchers have been mainly interested in "single"
proteins up until now, protein "marriages" still remain a mystery.
New light has just been shed on this area of biology thanks to biophysicists
at the Institut Curie. Using atomic force microscopy, Jean-Louis Rigaud's
team has been able to visualize the assembly of two membrane proteins
for the first time: images which provide information about the organization
of the protein complex as well as about the role it plays.
This advanced imaging technique should soon be used to answer other questions,
particularly those dealing with cell signaling, the prime example of the
interaction between proteins and whose malfunction is at the root of carcinogenesis.
This research is published in the Proceedings of the National Academy
of Sciences of February 18, 2003.
The organism is made up of
a multitude of organelles that play different roles. In order for them
to take their proper place, they must be differentiated and, above all,
isolated. This is the role of membranes. Made up of a double layer of
lipids, membranes surround the cells and thus limit their volume.
But membranes are not just simple walls. They are also border guards.
And this is where membrane proteins come in. It is precisely these proteins
that filter the passage between the two environments. As a result, they
play a key roll in cell signaling, a basic function of cell life and which,
if changed, can have extremely dire consequences for the cell and even
for the organism as a whole. The accumulation of defects in several genes
involved in the transmission of signals and the monitoring of the enzyme
apparatus is responsible for setting off the process of carcinogenesis
(see box).
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Cancer,
a signal transmission disease
Cells
are not isolated entities: they are in constant communication with
their environment thanks to membrane receptors containing informative
messages from the outside (other cells, tissues and organs). When
these messages are received, the proteins within the cell are activated
and then activate others which activate others, and so on. Once
these signals have been interpreted, they enable the cells to determine
their position and their role in the organism. They are absolutely
essential for cell proliferation, differentiation, morphology and
mobility. These signals are responsible for the harmonious balance
of the size and the function of tissues at the organ level.
The slightest malfunction of this system can lead to disaster: cancerous
cells, even if they no longer fulfill their role, proliferate in
the organism. They "ignore" orders from the outside, making
cancer, among others, a signal transmission disease.
The first "anti-signaling" drugs (Herceptin®, Iressa®
and Glivec®) have already been developed.
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Proteins that don't like
to be alone
Membrane proteins are extremely numerous since approximately 25% of the
sequenced genomes encode such proteins. They generally do not function
alone but in assemblies that form multi-protein complexes. One of the
most well known as of this time is the one responsible for transforming
light energy into ATP* in photosynthetic bacteria such
as Rhodopseudomonas viridis (see box). Although knowledge of the
different elements making up these membranes at the atomic level is relatively
advanced, very little information is available on the organization and
the function of these supramolecular complexes. Until now, this research
was a challenge for biologists since available methods only made it possible
to observe proteins isolated from their context.
Jean-Louis Rigaud's team, "Cristallisation bidimensionnelle de protéines
membranaires"**, at the Institut Curie, has just
changed all this by making high-resolution imaging of biological membranes
under physiological conditions possible using atomic force microscopy
(AFM).
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A
model photosynthetic bacteria
Photosynthesis
makes it possible to convert light energy into chemical energy that
can be used by the cell. This operation takes place at the level
of specialized membranes and requires several membrane proteins:
light trapping complexes, electron transport chains and ATPase.
The use of bacteria as study models rather than plants offers a
great number of advantages: the possibility of quickly obtaining
stable biological material in large quantities and the knowledge
of the atomic structures of all of the membrane constituents. However,
the way it works and the organization of the complex is not known.
This is especially the case with "core-complexes," where
the first stages of photosynthesis take place and which are made
up of light harvesting complexes (LH1) consisting of 16 sub-units
surrounding a reaction center (RC), formed by four sub-units.
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AFM or how to "pet"
proteins
Atomic force microscopy, developed by physicists in 1986, allows the imaging
of the surface of a sample at atomic resolution. The principle consists
of scanning the surface of the sample with a tip whose movements are recorded
by a laser. A "topographic" map of the sample can then be established
on the basis of these data.
AFM offers enormous benefits for analyzing samples in solution, a major
advantage for biology. By 1995, it was possible to observe membrane proteins
using AFM at lateral resolutions of 6 angstroms (10-10m) and
vertical resolutions of 1 angstrom. Until now, this approach was mainly
used for purified and reconstituted proteins in the form of 2D crystals
in double layers of lipid molecules. However, as a result of its precision,
AFM now makes it possible to visualize individual proteins in a membrane.
The problem thus becomes one of being able to get the AFM tip close enough
to the proteins to be able to gather the maximum amount of information
without damaging them – a delicate sleight of hand and one that Jean-Louis
Rigaud's team excels at. Researchers at the Institut Curie recently observed
the "relief" of isolated membrane proteins without prior crystallization.
They were therefore able to study conformation changes related to the
loss of only 20 amino acids of a protein with molecular precision (on
the order of 10 angstroms).
Jean-Louis Rigaud's team has just taken a major step by observing protein
interactions in native membranes, that is, those close to their natural
state. These are the first images of the organization of a multi-protein
complex.
Researchers at the Institut Curie are not just satisfied to be simple
observers. They modify the complex by specifically "tearing it off"
the protein sub-units with the AFM tip. The combination of imaging and
of this nanodissection makes it possible to obtain a detailed description
of the protein complex, leading to a better understating of the interaction
between the two types of proteins present.
In addition to a better understanding of photosynthesis in bacteria, these
results illustrate the advantage of using AFM to observe proteins at the
atomic level in native membranes. Thanks to AFM, Simon Scheuring, a member
of Jean-Louis Rigaud's team, introduces us to the world of proteins by
observing them in a multi-protein complex in situ and under physiological
conditions. Researchers at the Institut Curie are now concentrating on
more complex protein membrane assemblies.
It is therefore thanks to the combination of high-resolution microscopy
like AFM, electron microscopy – from atomic levels obtained by crystallography
all the way to the cell level – and optical microscopy, that cells
will be able to gradually reveal their secrets.
*ATP: molecule transporting energy into cells.
**Joint Research Unit 168, CNRS/Institut Curie, "Physicochimie
Curie," under the supervision of Jean-François Joanny.
Reference
Nanodissection and high-resolution imaging of the Rps. Viridis photosynthetic
core-complex in native membrane by AFM.
Simon Scheuring(1), Jérôme Segin(2), Sergio Marco(1),
Daniel Lévy(1),
Bruno Robert(2)
and Jean-Louis Rigaud(1)
Proceedings of the National Academy of Sciences (www.pnas.org),
vol. 100, pp.1690-93, February 18, 2003.
(1) JRU 168, "Physicochimie Curie", CNRS, and
LRC-CEA/Institut Curie.
(2) Service de Biophysique des Fonctions Membranaires, Département
de Biologie, Joliot-Curie, CEA, URA 2096.
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