New ways to probe chemical structure & dynamics using multi-frequency pulsed EPR

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I propose a novel methodology, using time domain, multi-frequency EPR to determine distances andorientation between a nitroxide spin label and a fast relaxing paramagnetic metal ion within a complex molecule like polymer or protein. This can be achieved by measuring the longitudinal relaxation time, T1 , of a spin label at different fields, which is enhanced by dipole-dipole coupling with the fast relaxing anisotropic paramagnetic metal centre. The procedures will be applicable both at low and room temperature conditions and alsowill enable analysis of complex motional dynamics of both the spin label and a molecular domain. The different types of motional dynamics of spin label and molecules will be modelled using the method of Brownian dynamics trajectories and a general computer simulation program will be designed. Validation will be carried out by measurement of longitudinal spin relaxation times at different frequencies from chemical and biological systems of known structure labelled to provide a set of distances and angels between a spin label and a rapidly relaxing metal centre including lanthanide(III) ions for ultra-fast relaxation. Lanthanide complexes covalently attached to a cystein residue will allow determination of distance-angles between two differently labelled proteins. Methodology developed will be applied at the Metallobiology Centre at UEA where new experimental opportunities have open up with the installation of a Bruker hybrid X/W-band Fourier Transform, pulsed EPR instrument, unique so far in the UK. New methods would open up the possibility for applications to various systems from liquid and molecular crystals and polymers to proteins in membrane and in the whole cell.