Clinical Exchange in a RNA-binding Protein

Dynamic properties of proteins are closely related to their function. Concerning elucidation of specificity and affinity of proteins to their interaction partners, NMR spectroscopy, in terms of nuclear spin relaxation measurement, has become an increasingly important tool in order to characterise local flexibility in proteins. This project concentrates on dynamics on a micro-s to ms time-scale, referred to as chemical/conformational exchange, that modulates the characteristic Larmor precession of a given nucleus by reversibly transferring it between different magnetic environments. This leads to an additional contribution to transverse relaxation rates. Analysis will focus on CPMG-type experiments that provide insight into underlying processes in terms of equilibrium populations, the exchange rate constant, and the chemical shift differences of a given nucleus between the different sites. Such experiments will be carried through to characterise exchange events in Gln and Asn side chain NH2 groups as well as in CH3 groups of Met residues. Human ribonuclear protein U1A is an RNA binding protein. It forms a trimolecular high-affinity complex with a second molecule U1A and its polyadenylation inhibition element (PIE, part of the untranslated region of its cognate m-RNA). This complex inhibits polyadenylation of m- RNA by binding to the enzyme polyadenylate polymerase (PAP). Exchange measurements will be carried out on free U1A (residues 2- 102) and U1A complexed to one high-affinity site of its PIE. Ps/ns dynamics measurements in backbone 15N and side chain CH2D methyl moieties have already indicated the presence of chemical exchange in sites coincident with residues involved in ligand interaction. Information on chemical exchange will provide further insight into the causes for the high affinity and specificity of the interactions.