| Popis: |
Pulse Dipolar Electron Paramagnetic Resonance (PD-EPR) spectroscopy, has become a very powerful method for investigating the structures and conformational equilibria biological systems adapt during their function. Pulse ELectron-electron DOuble Resonance (PELDOR) spectroscopy, can recover the weak magnetic dipole-dipole coupling interaction between paramagnetic centres. The latter ones are often endogenous to the system, like paramagnetic metal ions, protein bound radical cofactors, iron sulfur clusters, or amino acid radicals. Nevertheless, with the invention of the Site-Directed Spin-Labelling (SDSL), it has become possible to covalently bind a spin label to a cysteine residue engineered to a specific site of a protein. In combination with PD-EPR, this method has been extensively used for studying the structures of biomolecules, conformational changes, conformational states and multimerisation equilibria, by probing different sites of the molecules through spin labelling. Here, a method using PELDOR to study conformational changes within one monomeric unit of a multimeric system is proposed. This consists in identifying the intra-monomer distance among the inter-monomer distances when the monomers are doubly spin labelled. Moreover, a solution multimerisation study of a model protein found as dimer in its crystal structure is presented. The Relaxation-Induced Dipolar Modulation Enhancement (RIDME) experiment, is another method to resolve weak dipole-dipole interactions between paramagnetic centres. It has become a very attractive alternative to PELDOR when the measurements involve paramagnetic metal ions, because the dipolar interaction is induced by the spontaneous longitudinal relaxation of the metal ion spins which often display broad spectra limiting PELDOR sensitivity. An approach to retrieve angular constraints from RIDME distance measurements is shown. This is performed on a protein model system using a copper(II)-nitroxide spin pair. Finally, a metal templated dimerisation model is demonstrated to simultaneously match PELDOR and RIDME data for monitoring and characterising the dimerisation of monomeric units of a protein on a template. "I gratefully acknowledge EaStCHEM, School of Chemistry University of St Andrews, and the Engineering and Physical Sciences Research Council (EPSRC) for the financial support." -- Acknowledgements |
