title: Gas-Phase FRET Efficiency Measurements To Probe the Conformation of Mass-Selected Proteins.
abstract: Electrospray ionization and mass spectrometry have revolutionized the chemical analysis of biological molecules, including proteins. However, the correspondence between a protein's native structure and its structure in the mass spectrometer (where it is gaseous) remains unclear. Here, we show that fluorescence (Förster) resonance energy transfer (FRET) measurements combined with mass spectrometry provides intramolecular distance constraints in gaseous, ionized proteins. Using an experimental setup which combines trapping mass spectrometry and laser-induced fluorescence spectroscopy, the structure of a fluorescently labeled mutant variant of the protein GB1 was probed as a function of charge state. Steady-state fluorescence emission spectra and time-resolved donor fluorescence measurements of mass-selected GB1 show a marked decrease in the FRET efficiency with increasing number of charges on the gaseous protein, which suggests a Coulombically driven unfolding and expansion of its structure. This lies in stark contrast to the pH stability of GB1 in solution. Comparison with solution-phase single-molecule FRET measurements show lower FRET efficiency for all charge states of the gaseous protein examined, indicating that the ensemble of conformations present in the gas phase is, on average, more expanded than the native form. These results represent the first FRET measurements on a mass-selected protein and illustrate the utility of FRET for obtaining a new kind of structural information for large, desolvated biomolecules.
authors.affiliation: §Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Czar
authors.affiliation: †Biochemisches Institut, Universität Zürich, Zürich, CH-8057, Switzerland.
authors.forename: Franziska
authors.initials: F
authors.lastname: Zosel
authors.affiliation: †Biochemisches Institut, Universität Zürich, Zürich, CH-8057, Switzerland.
authors.forename: Iwo
authors.initials: I
authors.lastname: König
authors.affiliation: †Biochemisches Institut, Universität Zürich, Zürich, CH-8057, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettels
authors.affiliation: †Biochemisches Institut, Universität Zürich, Zürich, CH-8057, Switzerland.
authors.forename: Bengt
authors.initials: B
authors.lastname: Wunderlich
authors.affiliation: †Biochemisches Institut, Universität Zürich, Zürich, CH-8057, Switzerland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuler
authors.affiliation: †Biochemisches Institut, Universität Zürich, Zürich, CH-8057, Switzerland.
authors.forename: Arash
authors.initials: A
authors.lastname: Zarrine-Afsar
authors.affiliation: §Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Jockusch
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D005740:Gases / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D005740:Gases; D011506:Proteins