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permanent staff :
Karen Perronet, Nathalie Westbrook, Philippe Bouyer
members :
David Dulin, Antoine Le Gall
Collaborators at Institut de Chimie des Substances Naturelles (CNRS, Gif-sur-Yvette) :
Satoko Yoshizawa, Dominique Fourmy, Nicolas Soler
1. Biological context (return to top of page)
We are interessed in understanding how proteins are synthetized in cells. The proteins are molecules made of amino acids. The genetic code for all proteins is contained in DNA: 3 nucleotides code for 1 amino acid. In order to make a protein, the code is first written up on a messenger RNA (mRNA). After, the mRNA codons have to be associated with the amino acids. The molecular motor able to read the codons is the ribosome. It moves along the mRNA, and associates each codon to the matching anti-codon of a transfer RNA (tRNA) charged with the corresponding amino-acid. The ribosome also catalyses the formation of a peptidic bond between the following amino acids and thus translates the protein from the mRNA.
The motions of the ribosome subunits, of the proteic factors and of the substrates are essential to the biosynthesis of proteins. Our goal is to develop advanced optical methods of analysis at the single molecule level to understand the structural bases of these motions and in particular of the translocation process, motion of the transfer RNA-messenger RNA complex (tRNA-mRNA) within the ribosome. These motions are asynchronous and occur along multiple kinetic paths, thus a study on the single macromolecule level is particularly well suited. In collaboration with a team of biologists at ICSN, we are implementing a combination of different techniques - optical tweezers using laser beams, fluorescence imaging, fluorescent resonant energy transfer (FRET) - to study this reading mechanism of the genetic code.
From DNA to protein: the role of the ribosome. return to top of page
2. Physical tools (return to top of page)
2.1 Detection of single fluorescent molecules (return to top of page)
Total internal reflection fluorescence microscopy
The biomolecules we are studying are very small. Even the ribosome has a typical size of 20 nm. It is thus impossible to see them with usual optical microscopes. In order to localise these molecules, we label them with fluorescent markers (molecules or semi-conductor nano-crystals) that we can see by fluorescence microscopy. We developed a setup of total internal reflection fluorescence microscopy (TIRFM). We use an evanescent wave to excite the fluorescence of markers attached to a microscope slide. To do so, we use an oil-immersion objective with a very high numerical aperture (1.45 in our case) to make the excitation laser arrive on the slide with an angle higher than the total internal reflection critical angle. The evanescent wave generated on the slide decays within typically 100 nm in the sample. This plays the role of spatial selection (comparable with the pinhole used for confocal microscopy setups): only the molecules located on the slide can be excited. The fluorescence of the molecules is collected with the same objective and sent on a CCD camera. If the molecules are enough spaced on the slide, it is possible to detect the signal coming from one single molecule. The advantage of the TIRFM setup compared to confocal geometries is that, at the same time, we collect the signal of many single molecules.
A good marker for amino acids: BODIPY-FL
Setup for total internal reflection fluorescence microscopy.
We used this setup to study one particular fluorescent molecule: BODIPY-FL. In spite of its photobleaching, this small fluorophore is potentially useful for monitoring biochemical processes like protein synthesis because it can be efficiently incorporated into proteins by the ribosome through a charged epsilon-labeled fluorescent lysine tRNA. Using evanescent wave laser excitation at 488 nm, we have been able to observe single Bodipy molecules using integration times as low as 20 ms with a good signal to noise ratio, and for several images before photobleaching. We have measured the fluorescence decay due to photobleaching and have been able to lengthen it by a factor of 5 using oxygen scavenger systems.
Experimental results for BODIPY-FL single molecules observed with TIRFM. BODIPY-Fl single-stranded DNA are linked to the slide through biotin/streptavidin linkage. We show an image of several single BODIPY-FL molecules observed in TIRFM. This image is the first of a stack of 25 images taken of the same area of the sample. About 180 single molecules can be identified in this image, and their photobleaching times are computed in the histogram (top right). The second histograms (bottom right) show that it is possible to increase the BODIPY-FL lifetime before photobleaching by removing the oxygen from the sample. This was done by adding an oxygen scavenger system to the sample buffer.
Setup for multi-color total internal reflection fluorescence microscopy. Different excitation lasers are available on our setup to co-localize different types of fluorescent markers fixed to the different biological species of our study.
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2.2 Manipulation of single molecules: optical tweezers (return to top of page)
In the 70's, Ashkin demonstrated the possibility to trap a dielectric object with a laser beam. Since, this technique is widely used, in particular to study biological motors. It is now possible to measure with a excellent accuracy forces in the 1-100 pN range, which is typically the forces exerted by bio-motors.
Principals of optical trapping
A dielectric object in a laser beam has two forces exerted on it. The radiation pressure, due to the photons reflecting on the object, pushes it in the direction of light propagation. But there is also a force proportional to the intensity gradient, which is due to the refracted photons. These force will bring the object at the highest intensity point. If the intensity gradient is high enough, it is thus possible to trap the object. We use a high numerical oil-immersion objective (the same as the one used for TIRFM) to tightly focus the trapping laser. We chose a Nd:YAG (1064 nm) laser because its wavelength is in the transparence window of biological media (water, hemoglobin).
Force measurement
To be able to measure the force exerted on a trapped bead, we record the location of this bead in the trap. If the bead is near the center of the trap, we can assume the trap is harmonic and knowing its stiffness, we get the force.
Picture of the optical tweezers setup.
Scheme of the force measurement system.
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3. Current projects (return to top of page)
Kinetics of amino-acids incorporation in a growing protein:
We are interested in measuring the kinetics of incorporation of amino acids in a protein being translated by a ribosome. We use TIRFM to record the arrival time of labelled amino acids in the growing protein.
Translation of selenoproteins:
We are studying the interactions between different factors (proteins, mRNA structures) involved in the translation of selenoprotein. We try to characterize the force of these interactions using optical tweezers and the dynamics of conformational changes with resonant energy transfer measurements.
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Offres de stages, thèses et postdocs (return to top of page)
All offers of the Atom Optics group
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