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Please use this identifier to cite or link to this item: http://hdl.handle.net/2262/65711

Title: Effect of the point mutation H148G on GFPmut2 unfolding kinetics by fluorescence spectrocopy
Keywords: GFP
unfolding
fluorescence anisotropy
fluorescence correlation spectroscopy
(H148G
GFPmut2)
Issue Date: 30-May-2011
Publisher: Elsevier
Abstract: 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.
Graphical abstract
URI: http://hdl.handle.net/2262/65711
ISSN: 03014622 (ISSN)
DOI: 10.1016/j.bpc.2011.04.004
Rights: 2010
Affiliation: Dipartimento di Fisica, Università degli Studi di Milano-Bicocca - Piazza della Scienza 3--> , 20126 Milano--> - ITALY (Bosisio, Chiara)
Laboratorio di Biologia Molecolare - Scuola Normale Superiore--> , Pisa--> , 50126--> - (Quercioli, Valentina)
Dipartimento di Fisica, Università degli Studi di Milano-Bicocca - Piazza della Scienza 3--> , 20126 Milano--> - ITALY (Chirico, Giuseppe)
Dipartimento di Fisica, Università degli Studi di Milano-Bicocca - Piazza della Scienza 3--> , 20126 Milano--> - ITALY (D'Alfonso, Laura)
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma - Viale Usberti 23/A--> , 43124 Parma--> - ITALY (Bettati, Stefano)
Istituto Nazionale di Biostrutture e Biosistemi - Rom--> - ITALY (Bettati, Stefano)
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma - Viale Usberti 23/A--> , 43124 Parma--> - ITALY (Raboni, Samanta)
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma - Viale Usberti 23/A--> , 43124 Parma--> - ITALY (Campanini, Barbara)
Dipartimento di Fisica, Università degli Studi di Milano-Bicocca - Piazza della Scienza 3--> , 20126 Milano--> - ITALY (Collini, Maddalena)
ITALY (Collini, Maddalena)
Appears in Collections:PEER Publications

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