Advanced Developments of Electron Spin Labeling as High-Resolution Sensors of Protein Structure and Conformational Switching

摘要

The dynamic modes of a nitroxide side chain in a protein directly reflect both the local 3D structure and dynamics of the protein. If the structural and dynamical contributions can be resolved, a map of side chain mobility throughout a protein molecule can provide an image of protein structure and dynamic features related to function. To separate these contributions, libraries of spin-labeled mutants of T4 lysozyme (T4L), Myoglobin (Myb) and Cellular Retinol Binding Protein (CRBP) were prepared, and the corresponding EPR spectra analyzed by simulation techniques. In addition, x-ray structures were determined for select spin-labeled T4 mutants. The collective results of these efforts have provided an unprecedented level of sophistication in interpretation of the EPR spectra of labeled proteins, and establish the feasibility of separating structural and dynamical contributions. In addition, a 'bridge project' to the DARPA MOSAIC project has been completed. In this venture, spin labeled Annexin XII and T4L were supported on planar surfaces and oriented in the external magnetic field of the EPR spectrometer. Such specimens provide an additional dimension (orientation) to extract structural information, and provide the basis for producing suitable specimens for the single spin microscope under construction at UCLA.