Time-resolved NMR spectroscopy: ligand-induced refolding of riboswitches.

A detailed understanding of cellular mechanisms requires knowledge of structure and dynamics of the involved biomacromolecules at atomic resolution. NMR spectroscopy uniquely allows determination of static and dynamic processes at atomic level, including structured states often represented by a single state as well as by unstructured conformational ensembles. While a high-resolution description of structured states may also be obtained by other techniques, the characterization of structural transitions occurring during biomolecular folding is only feasible exploiting NMR spectroscopic methods. The NMR methodical strategy includes the fast initiation of a folding reaction in situ and the possibility to detect the induced process with sufficient time resolution on the respective NMR time scale. In the case of ligand-induced structural transitions of RNA, the initiation of the folding reaction can be achieved by laser-triggered deprotection of a photolabile caged ligand whose release induces folding of a riboswitch RNA. The strategy discussed here is general and can also be transferred to other biological processes, where at least one key reagent or substrate, e.g., ions, ligands, pH, or one specific conformational state, can be photochemically caged. The rates of reversible and irreversible reactions or structural transitions that can be covered by real-time NMR methods range from milliseconds up to hours.In this chapter, we discuss the application of a time-resolved NMR strategy to resolve the ligand-induced folding of the guanine-sensing riboswitch aptamer domain of the B. subtilis xpt-pbuX operon.