title: Action-Self Quenching: Dimer-Induced Fluorescence Quenching of Chromophores as a Probe for Biomolecular Structure.
abstract: To obtain a more detailed understanding of how structure influences the function and interaction of biomolecules, it is important to develop structure sensitive techniques to probe these relationships. Alongside in vivo and in vitro techniques, it is instructive to consider in vacuo methodologies: for example native mass spectrometry, ion mobility mass spectrometry, and FRET. Here, we propose a novel technique for probing biomolecular structure based on the changes in photophysics of a chromophore upon dimer formation. Comparison of solution and gas phase measurements on a doubly tagged tripeptide shows that dimer-induced fluorescence quenching is accompanied by an increase in photofragmentation yield. The 12-28 fragment of amyloid beta was used to show that as the charge state was increased-previously shown to cause a conformational change from compact random coil to extended helical structure-the disappearance of a band at 495 nm could be correlated with the level of self-quenching. The presence of features in the action spectrum of the +3 charge state of both quenched and unquenched chromophores allowed inference of multiple conformations. Single wavelength measurements on doubly tagged ubiquitin cations were performed to show that the technique is feasible on a small protein. These results demonstrate that self-quenching is a sensitive and fast gas-phase probe of biomolecular structure that can be directly linked to solution phase measurements. Further, it is capable of probing very small changes in conformation, making it complementary to FRET based techniques, which are insensitive at very short chromophore separations.
authors.affiliation: Université Lyon, Université Claude Bernard Lyon 1 , CNRS, Institut Lumière Matière UMR 5306, F-69100, Villeurbanne, France.
authors.forename: Steven
authors.identifier: 0000-0002-3268-8247
authors.initials: S
authors.lastname: Daly
authors.affiliation: Université Lyon, Université Claude Bernard Lyon 1 , CNRS, Institut Lumière Matière UMR 5306, F-69100, Villeurbanne, France.
authors.forename: Chang Min
authors.identifier: 0000-0001-7708-9056
authors.initials: CM
authors.lastname: Choi
authors.affiliation: Université Lyon, Université Claude Bernard Lyon 1 , Ens de Lyon, CNRS, Institut des Sciences Analytiques UMR 5280, 5 rue de la Doua, F-69100, Villeurbanne, France.
authors.forename: Fabien
authors.identifier: 0000-0001-9115-9233
authors.initials: F
authors.lastname: Chirot
authors.affiliation: Université Lyon, Université Claude Bernard Lyon 1 , CNRS, Institut Lumière Matière UMR 5306, F-69100, Villeurbanne, France.
authors.forename: Luke
authors.initials: L
authors.lastname: MacAleese
authors.affiliation: Université Lyon, Université Claude Bernard Lyon 1 , CNRS, Institut Lumière Matière UMR 5306, F-69100, Villeurbanne, France.
authors.forename: Rodolphe
authors.identifier: 0000-0001-5682-8550
authors.initials: R
authors.lastname: Antoine
authors.affiliation: Université Lyon, Université Claude Bernard Lyon 1 , CNRS, Institut Lumière Matière UMR 5306, F-69100, Villeurbanne, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Dugourd
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D005740:Gases / Q000737:chemistry; D000075663:Ion Mobility Spectrometry; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary
chemical_list: D016229:Amyloid beta-Peptides; D005456:Fluorescent Dyes; D005740:Gases; D009842:Oligopeptides