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EPR Spectroscopy; Protein-Lipid Interactions
Abstract:
Site-directed spin labeling (SDSL) has proven to be a powerful tool for the investigation of structure and dynamics of proteins. SDSL involves introduction of nitroxide side chains (R1) at selected positions of the protein sequence, followed by an analysis of the EPR line shape to deduce the secondary and tertiary structure. The examination of protein structure and dynamics at interfaces is an important application of this technique. The information content can be increased if ordered arrays of protein molecules are investigated. These arrays were prepared by tethering histidine-tagged T4 Lysozyme (T4L) to a single planar supported lipid bilayer on quartz via chelating headgroups (Ni-NTA). For molecules oriented in two dimensions, the tensorial nature of the Hamiltonian gives rise to angular dependent EPR spectra which can be used to determine the orientation of the nitroxide relative to the surface. Such information is important for determining the topography of proteins bound to surfaces. In addition, direct information on the structure and interactions of the protein at the surface is obtained from the dynamics of the side chains inferred from the spectral line shape. Results for R1 residues at several sites in oriented monolayers of T4L will be discussed with respect to these points.
