EPR and NMR spectroscopy of spin-labeled proteins

Spin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in frozen solution were studied using 275 GHz EPR (J-band), with the advantage compared to 9 GHz (X-band) and 95 GHz (W-band) to allow differentiating between sites having small differences in polarity. The polarity/proticity profiles of the four mutants have been obtained and compared with data already reported in literature of spin labels in different solvents. In order to detect distances at the nm scale, azurin was used as a known model system for double electron-electron spin resonance. The conformation of the spin label linker was modelled. The model was found to be in good agreement with experimental results and can be extended to other proteins. The dynamics of the transient complex of Nostoc sp. PCC 7119 cytochrome f __ plastocyanin was investigated by NMR using the paramagnetic relaxation enhancements. The experimental distance restraints, used in docking calculations, are best interpreted by the presence of a dynamic ensemble of protein-protein orientations within the complex, rather than by a single, well-defined structure, and can be described with an encounter complex model.LEI Universiteit LeidenBiological and Molecular Physic

The Complex of Cytochrome f and Plastocyanin from Nostoc sp. PCC 7119 is Highly Dynamic

9Biological and Molecular Physic

Novel approaches for distance determination by EPR: large anisotropy, fast relaxing paramagnetic centres, such as Fe(III), as markers

Macromolecular BiochemistryBiological and Soft Matter Physic

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process. © 2014 Elsevier B.V.Peer Reviewe