Untersuchung der Struktur und Dynamik von T4 Lysozym auf planaren Oberflächen mittels ESR-Spektroskopie

An understanding of the interaction between proteins and surfaces is not only important for many biological queries but also of great technological interest. In studies ranging from the investigation of basic cell physiological mechanisms to the development of medical implants the interplay between the structure of adsorbed proteins and the surface properties are in the center of interest. However, this information is difficult to access, as the number of adequate experimental techniques to study the structure of adsorbed proteins on a molecular level is very limited. Introducing specific labels into proteins provides inherently a molecular spatial resolution regardless of the dimension of the system under investigation. Thus, the Site-directed Spin Labeling (SDSL) technique within the Electron Paramagnetic Resonance (EPR) Spectroscopy has evolved to a valuable technique to study the structure and dynamics of proteins on the level of the backbone fold. In this work the application of the SDSL technique has been extended to the analysis of proteins adsorbed to planar surfaces, in order to elucidate the influence of model surfaces of different physical and chemical properties on the structure and dynamics of the proteins. Specifically, the conformational changes the small globular model protein T4 Lysozyme undergoes in contact with quartz-supported lipid bilayers were in the center of investigation. By introducing spin-labeled lipids the model membranes were characterized simultaneously and protein-induced changes were monitored. In a first step a monolayer of spin-labeled T4 Lysozyme was tethered via a selective linker to a zwitterionic lipid bilayer. A quantitative line shape analysis of the EPR spectra reveals already minor tertiary rearrangements whereas the secondary structure is conserved. Moreover, angular dependent EPR spectra show the existence of an ordered array of adsorbed proteins. An analysis of the spectra in terms of protein orientation on the surface is developed. With stronger protein-surface interactions present structural changes of the protein can already be elucidated by qualitative line shape analysis. Thus, a partial unfolding of the small two-domain protein T4 Lysozyme as interacting with a negatively charged quartz surface is observed. The unfolding N-terminal domain is sticking to the surface whereas the C-terminal domain remains almost intact. The situation differs substantially, if T4 Lysozyme is adsorbed to a negatively charged model membrane. Here, the EPR spectra of the adsorbed protein suggest the formation of three-dimensional aggregates of structural modified proteins on the membrane surface. The fluidity of the model membrane is reflected in an structural answer of the model surface to protein adsorption in terms of a phase separation of the lipid mixture due to an accumulation of negatively charged lipids. Consequently, the Site-directed Spin Labeling technique proves to be a very useful tool to study the structure and dynamics of the protein-surface interfacial system. It is demonstrated, that the line shape analysis of the EPR spectra provides a detailed insight into the molecular interaction and adsorption mechanisms.