title: The AdoCbl-riboswitch interaction investigated by in-line probing and surface plasmon resonance spectroscopy (SPR).
abstract: The unique feature of riboswitches is to control selected gene expression by specific recognition of a cognate ligand. AdoCbl (adenosyl cobalamin, coenzyme B12) riboswitches regulate the expression of enzymes and transporters involved in bacterial AdoCbl biosynthesis or uptake on a transcriptional and/or translational level. The analysis of ligand recognition and the induced conformational changes requires a detailed knowledge of the kinetics and thermodynamics of ligand binding and of the secondary structure rearrangements. This chapter describes the investigation of coenzyme B12 binding to the btuB riboswitch from Escherichia coli by in-line probing assays and surface plasmon resonance spectroscopy. The experimental conditions, requirements, and performance of both methods are presented together with the evaluation of the experimental data to determine the associated conformational changes of the RNA and the kinetic and thermodynamic parameters of ligand binding. Owing to the light sensitivity of the cobalt(I)'carbon bond, these methods were specifically modified to ensure the chemical integrity of AdoCbl.
authors.affiliation: Department of Chemistry, University of Zurich, Zürich, Switzerland.
authors.forename: Michelle F
authors.initials: MF
authors.lastname: Schaffer
authors.affiliation: Department of Chemistry, University of Zurich, Zürich, Switzerland.
authors.forename: Pallavi K
authors.initials: PK
authors.lastname: Choudhary
authors.affiliation: Department of Chemistry, University of Zurich, Zürich, Switzerland. Electronic address: roland.sigel@chem.uzh.ch.
authors.forename: Roland K O
authors.initials: RK
authors.lastname: Sigel
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003038:Cobamides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058928:Riboswitch; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003038:Cobamides; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; D058928:Riboswitch; C000913:cobamamide
keywords: Coenzyme B12/AdoCbl; In-line probing; Ligand binding kinetics; RNA structure analysis; Riboswitch; SPR