title: Effect of the point mutation H148G on GFPmut2 unfolding kinetics by fluorescence spectroscopy.
abstract: We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins.
authors.affiliation: Dipartimento di Fisica, Università degli Studi di Milano-Bicocca, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Bosisio
authors.forename: Valentina
authors.initials: V
authors.lastname: Quercioli
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Chirico
authors.forename: Laura
authors.initials: L
authors.lastname: D'Alfonso
authors.forename: Stefano
authors.initials: S
authors.lastname: Bettati
authors.forename: Samanta
authors.initials: S
authors.lastname: Raboni
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.forename: Maddalena
authors.initials: M
authors.lastname: Collini
mesh_terms: D000596:Amino Acids; D005973:Glutamine / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; D005973:Glutamine; D049452:Green Fluorescent Proteins; D006639:Histidine