Probing Protein-RNA Interactions Through Spin-Labelling and Paramagnetic Relaxation Enhancements
Paramagnetic relaxation enhancement (PRE) methods can provide invaluable long range distance restraints well beyond those determined from NOEs. The versatility of the PRE approach has evolved from, almost exclusively, studies of metal-binding proteins, to a wide range of applications. This has been largely through the development of chemical approaches to the selective conjugation of non-metal based stable spin-labels to proteins through the use of site-directed mutagenesis, or through the nucleobase of a 5-thiouridine (RNA) via solid-phase synthesis. The introduction of a spin-label within a sequence of nucleic acids has the potential to identify the position and relative orientation of protein recognition motifs arranged along the RNA chain. We have used the selective spin-labelling of RNA to probe interactions with the CUG-BP, Elav-like family (CELF) of RNA-binding proteins which control gene expression by regulating pre-mRNA splicing, deadenylation and mRNA stability. We demonstrate how PRE effects on the 1H-15N-TROSY spectrum of a 320 residue three-domain protein construct identified a specific and non-sequential arrangement of binding motifs along the RNA target sequence. Analogous site-selective spin-labelling methodologies, combined with NMR analysis, is widely applicable to a range of challenges in structural biology in determining complex biomolecular assemblies and dynamics.
KeywordsChemical shift perturbations Paramagnetic relaxation enhancement Spin-labelling approach Protein-RNA interactions
- 1.Wuthrich K. NMR of proteins and nucleic acids. New York: Wiley; 1986.Google Scholar
- 9.Neuhaus D, Williamson MP. The nuclear Overhauser effect in structural and conformational analysis. 2nd ed. New York: Wiley-VCH; 2000.Google Scholar