title: ITC analysis of ligand binding to preQ₁ riboswitches.
abstract: Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, K(A), of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.
authors.affiliation: Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Liberman
authors.affiliation: Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Jarrod T
authors.initials: JT
authors.lastname: Bogue
authors.affiliation: Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Structural Biology & Biophysics Facility, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Jermaine L
authors.initials: JL
authors.lastname: Jenkins
authors.affiliation: Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Salim
authors.affiliation: Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Structural Biology & Biophysics Facility, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. Electronic address: joseph.wedekind@rochester.edu.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Wedekind
mesh_terms: D002151:Calorimetry / Q000379:methods*; D008024:Ligands; D008958:Models, Molecular; D011744:Pyrimidinones / Q000737:chemistry / Q000378:metabolism*; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism*; D058928:Riboswitch; D013816:Thermodynamics
chemical_list: C096876:7-(aminomethyl)-7-deazaguanine; D008024:Ligands; D011744:Pyrimidinones; D011758:Pyrroles; D058928:Riboswitch
keywords: Crystallography; Gene regulation; ITC; K(D); Ligand binding; PreQ(1); Riboswitch; Thermodynamic analysis