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Motivation

Molecular recognition plays a pivotal role in nature. Signalling cascades, enzymatic activity, genome replication and transcription, cohesion of cellular structures, interaction of antigens and antibodies and metabolic pathways all rely critically on specific recognition. In fact, every process in which molecules with each other in a specific manner depends on this trait. Molecular recognition studies emphasize specific interactions between receptors and their cognitive ligands. Despite a growing body of literature on the structure and function of receptor-ligand complexes, it is still not possible to predict reaction kinetics or energetics for any given complex formation, even when the structures are known. Additional insights, in particular about the molecular dynamics within the complex during the association and dissociation process, are needed. The high-end strategy is to probe the energy landscape that underlies the interactions between molecules, whose structure is known at atomic resolution.
Receptor-ligand complexes are usually formed by a few, non-covalent weak interactions between contacting chemical groups in complementary determining regions, supported by framework residues providing structurally conserved scaffolding. Both the complementary determining regions and the framework have a considerable amount of plasticity and flexibility, allowing for conformational movements during association and dissociation. In addition to knowledge about structure, energies, and kinetic constants, information about these movements is required for the understanding of the recognition process. Deeper insight into the nature of these movements as well as the spatiotemporal action of many weak interactions, in particular the cooperativity of bond formation, is the key for the understanding of receptor-ligand recognition.
For this, experiments at the single molecule level, on time scales typical for receptor-ligand complex formation and dissociation appear to be required. The potential for the atomic force microscope (AFM) (Binning et al. 1986) to measure ultra-low forces at high lateral resolution has paved the way for single molecule recognition force microscopy studies. The particular advantage of AFM in biology is that the measurements can be carried out in aqueous and physiological environment, which opens the possibility for studying biological processes in vivo. The core methodology described in this application for investigating molecular dynamics of receptor-ligand interactions, Molecular Recognition Force Microscopy (MRFM) (Lee et al. 1994a; Florin et al. 1994; Hinterdorfer et al. 1996), is based on scanning probe microscopy (SPM) technology (Binning et al. 1986). A force is exerted on a receptor-ligand complex and the dissociation process is followed over time. This requires a careful AFM tip sensor design, including tight attachment of the ligands to the tip surface. Dynamic aspects of recognition are addressed in force spectroscopy (FS) experiments, where distinct force-time profiles are applied to give insight into changes of conformations and states during receptor-ligand dissociation. MRFM is a key tool to explore kinetic and structural details of receptor-ligand recognition.
Likewise, the real time visualization and quantification of receptor binding sites on cell surfaces remains a fundamental challenging task in molecular cell biology. This can be achieved by common techniques such as immunostaining (or immunocytochemistry) or by sophisticated optical techniques such as STED microscopy (Willig et al. 2006), NSOM (Ianoul et al. 2004; Koopman et al. 2004) or single molecule optical microscopy (Baumgartner et al. 2003; Schmidt et al. 1996). The lateral resolution in these studies ranges from a few tens of nanometres to about 200 nm. Despite the fast time resolution, in optical studies no information about topography is attainable. In contrast, AFM, which represents a scanning microscopy technique, offers a unique solution to obtain topography images with nanoscale resolution. With the resent development of simultaneous Topography and RECognition (TREC) technique, it becomes possible to quickly and easily obtain maps of binding sites with the lateral accuracy of several nm across variety surfaces, as it has been demonstrated on model receptor-ligand pairs (Ebner et al. 2005; Stroh et al. 2004b) and remodelled chromatin structures (Stroh et al. 2004a). Although TREC has been very recently exploited in order to locally identify VE-cadherin binding sites on MyEnd cells and to colocalize their position with membrane topographical features (Chtcheglova et al. 2007), it appears highly profitable to develop a new generation of microscopes containing a rich combination of highly sensitive spectroscopy/microscopy methods and modes, to explore cellular systems of complex composition, organization and processing in space and time.