title: Evidence of discrete substates and unfolding pathways in green fluorescent protein.
abstract: We present evidence of conformational substates of a green fluorescent protein mutant, GFPmut2, and of their relationship with the protein behavior during chemical unfolding. The fluorescence of single molecules, excited by two infrared photons from a pulsed laser, was detected in two separate channels that simultaneously collected the blue or the green emission from the protein chromophore chemical states (anionic or neutral, respectively). Time recording of the fluorescence signals from molecules in the native state shows that the chromophore, an intrinsic probe sensitive to conformational changes, switches between the two states with average rates that are found to assume distinct values, thereby suggesting a multiplicity of protein substates. Furthermore, under denaturing conditions, the chromophore switching rate displays different and reproducible time evolutions that are characterized by discrete unfolding times. The correlation that is found between native molecules' switching rate values and unfolding times appears as direct evidence that GFPmut2 can unfold only along distinct paths that are determined by the initial folded substate of the protein.
authors.affiliation: Department of Physics, University of Milano-Bicocca, Milan, Italy. baldini@mib.infn.it
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Baldini
authors.forename: Fabio
authors.initials: F
authors.lastname: Cannone
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Chirico
authors.forename: Maddalena
authors.initials: M
authors.lastname: Collini
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.forename: Stefano
authors.initials: S
authors.lastname: Bettati
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017510:Protein Folding; D013379:Substrate Specificity
chemical_list: D049452:Green Fluorescent Proteins