title: smFRET studies of the 'encounter' complexes and subsequent intermediate states that regulate the selectivity of ligand binding.
abstract: The selectivity with which a biomolecule can bind its cognate ligand when confronted by the vast array of structurally similar, competing ligands that are present in the cell underlies the fidelity of some of the most fundamental processes in biology. Because they collectively comprise one of only a few methods that can sensitively detect the 'encounter' complexes and subsequent intermediate states that regulate the selectivity of ligand binding, single-molecule fluorescence, and particularly single-molecule fluorescence resonance energy transfer (smFRET), approaches have revolutionized studies of ligand-binding reactions. Here, we describe a widely used smFRET strategy that enables investigations of a large variety of ligand-binding reactions, and discuss two such reactions, aminoacyl-tRNA selection during translation elongation and splice site selection during spliceosome assembly, that highlight both the successes and challenges of smFRET studies of ligand-binding reactions. We conclude by reviewing a number of emerging experimental and computational approaches that are expanding the capabilities of smFRET approaches for studies of ligand-binding reactions and that promise to reveal the mechanisms that control the selectivity of ligand binding with unprecedented resolution.
authors: Department of Chemistry, Columbia University, New York, NY 10027, United States.
authors: Colin D
authors: CD
authors: Kinz-Thompson
authors: Department of Chemistry, Columbia University, New York, NY 10027, United States. Electronic address: rlg2118@columbia.edu.
authors: Ruben L
authors: RL
authors: Gonzalez
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008024:Ligands; D008853:Microscopy; D013379:Substrate Specificity
chemical_list: D008024:Ligands
keywords: Encounter complex; Fidelity; Ribosome; Single-molecule fluorescence resonance energy transfer; Spliceosome