Rasterkraftmikroskopische Untersuchungen an nativen biologischen Membranen und Sucroseporin als Beispiel eines rekonstituierten Membranproteins

Abstract

In the first part of this thesis methods for the immobilisation of biological objects on sufficiently smooth and clean surfaces were investigated. Appropriate substrates for scanning force microscopy (SFM) investigations are poly(vinyl phenyl ketone), PVPK, and several variants of furan polymers (FP). These materials allow both a functionalisation as specific cell culture substrate (PVPK, FP) and the variation of surface properties for the immobilisation of diverse membrane and protein preparations (FP). Thus, e.g. laminin can be adsorbed directly with a slightly increased roughness. Cells attach to this substrate in a mechanically stable fashion and show normal growth behaviour. The suitability of PVPK was shown with primary cultures and several cultured cell lines. Furan polymer substrates proved to be particularly suitable for the immobilisation of membrane preparations out of an aqueous suspension. Additionally, it could be shown that macromolecules can be adsorbed on modifications of FP.The reconstitution of Sucrose Porin with lipids and subsequent SFM-investigation represents the second part of this study. Both the surface exposed to the extracellular medium and the periplasmic surface of the protein could be imaged by SFM with submolecular resolution. The data was analysed using single particle averaging methods. Thus, it became possible to discriminate the electrolyte-exposed surfaces and to characterise domains of different flexibility. In the case of the periplasmic side, the more or less dominating influence of a flexible structure on the imaging process was observed. Depending on the applied force, however, the rigid periplasmic core of the beta-barrel trimer could be revealed as well. This is in good agreement with recent assumptions concerning the size and properties of an N-terminal coiled coil structure. On the extracellular surface, imaging was influenced by the force dependent contribution of loops exposed at the rim of the beta-barrel.