Further developpement of new single molecule force spectroscopy methods and their application to study the receptor - ligand interactions for viral surface proteins and inner nuclear proteins

New single molecule force spectroscopy methods and their application to study the receptor – ligand interactions for viral surface proteins and inner nuclear membrane proteins. The present project deals with the study of viral proteins, which are important when a virus infects a cell. The infection takes place when the surface of the virus attaches to the surface of the cell. The mechanism of this attachment depends upon the surface proteins of the virus and of the cell. By directly measuring with an Atomic Force Microscope the interaction between surface proteins of the cell and of the virus, we intend to indentify which proteins are responsible for the attachment and probably also for the infection of the cell by the virus.....Next

Scanning Near-Field Optical Microscopy - based study of local dynamics of receptor-ligand ineractions at the single molecule level
A protein is an amino-acid chain which has a specificactivity induced by its conformation [1]. In the cell, unfold or misfolded proteins are inactive or even plain toxic and can aggregate themselves into amyloid plaques well known in Alzjeimer's and other degenerative diseases. To limit these unfolded and aggregated protein, the 'molecular chaperones', some ATP dependant proteins, are present in the cell. Two famous chaperones are GroEL [2]and GroES. Their structure and functions are very well known, but their exact biochemical reaction cycle remains unsolved. For this reason, we dedicate a 'Scanining Near-field Optical Microscope' (SNOM) to study a real time this reaction cycle. Here is a principle: a very sharp optical fiber scans a sample where GroEL is bounded and GroES is in solution. During the scan, the topographic and optical image will be connected by an electronic device and informations about the reaction cycle will be collected. A special interest will be devoted to metastable states which are completly smeared out in bulk experiments, but can lead to important knowledge about the molecular conformational states of the complex.

Idea of FRET SNOM, which has been put forward around 1995 by Sekatskii and Letokhov [2], consists in fabrication of subwavelength-size local fluorescent probes which are excited by laser radiation and scanned at the contact (at a distance not exceeding a few nanometers) with the sample to be studied. When the distance between an appropriate donor/acceptor fluorescent center of the probe and an acceptor/donor fluorescent center of the sample is less than the characteristic Förster radius R0 of FRET for this particular pair, an excitation is effectively transferred to the acceptor which fluorescence can be detected by usual means. Due to the pseudocontact character of the FRET interaction (the acceptor’s excitation probability decreases as the sixth power of the donor – acceptor distance when it exceeds the value of R0), the spatial resolution for such an approach is governed not by the size of an aperture for the light transmission, as this is typical for apertured SNOM, but rather by the value of the R0, which for typical FRET pairs ranges 2 – 8 nm. Hence the resolution can be much improved in comparison with the value of 50 nm typical for standard apertured SNOM. .... Next