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2008 - Johannes Preiner

Fast Scanning AFM study of antibody-antigen binding dynamics on biological membranes

Dissertationsprojekt: Forschungsaufenthalt am Toshio Biophysics-Institute at the Kanazawa University, Kanazawa, Japan
22.10.2007 – 23.11.2007

Johannes Preiner
Contact: johannes.preiner(/\t)

            Binding geometry and structural changes invoked upon interaction between antibodies and membrane proteins are of considerable interest for a better understanding of the mechanisms of underlying receptor recognition and therefore important i n a variety of biological processes such as cell signalling and transmembrane signal transduction. The binding dynamics of the antibody-antigen interacti on is commonly expressed in kinetic parameters, on-rate and off-rate, usually quantified in ensemble average techniques. Owing to the high flexibility of the Fab and Fc fragment s, the two Fab fragments of an antibody can bind independently of each other. The kinetics of antibody binding to cell membr ane antigens is therefore determined by the inherent binding properties of the Fab fragment-antigen interaction, the flexibility and movement of the antibody molecule, and the lateral arrangement and mobility of the antigenic binding sites.

            The new Fast Scanning AFM developed by Toshio Ando (1-3) opens the possibility to observe the binding of antibodies with a high spatial resolution, and additionally a video rate- time resolution (~60 frames per second), i.e. one can follow the binding in realtime (so far, conventional AFM images were taken in the time-scale of minutes).

            During my stay in this lab, binding studies of IgG antibodies and other ligands on bacterial membr anes were performed using this new technique. The results revealed a highly dyna mic nature of all the samples investigated. In this way, diffusion of single membrane proteins within a lipid bilayer (PM), and the dynamic behaviour of single proteins arranged in two dimensional protein-lattices (S-layer) could be observed with sub- nanometer spatial and ~50 ms time resolution. The experiments revealed propagation of defects, rotations of unit cells and the flexibility of single sub-domains, which are important processes considering the self-assembling properties of these membranes. In addition, the dynamic behaviour of IgG span> antibodies upon binding to antigenic bindings sites embedded in membranes h ave been observed for the very first time, and a qualitative explanation of the origin of this dynamics could be given. Experiments involving ligands with other mechanical properties than IgGs, like Fab fragments of the very same antibodies and StrepTactin, were performed, supporting the given qualitative picture.

             In conclusion, novel insights into the dynamic behaviour of various biological samples were gained, which would not have been possible with any commercial ly available technique. From the scientific point of view, my stay in Japan w as a great success, additionally I learned a lot about the Japanese culture, and last but not least I’m quite experienced in using chop sticks now.