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Temperature–pressure stability of green fluorescent protein: A Fourier transform infrared spectroscopy study
Author(s) -
Scheyhing Carsten H.,
Meersman Filip,
Ehrmann Matthias A.,
Heremans Karel,
Vogel Rudi F.
Publication year - 2002
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.10237
Subject(s) - green fluorescent protein , fluorescence , chemistry , fourier transform infrared spectroscopy , mutant , infrared , biophysics , fluorescence spectroscopy , infrared spectroscopy , high pressure , spectroscopy , analytical chemistry (journal) , biochemistry , chromatography , chemical engineering , gene , organic chemistry , biology , thermodynamics , physics , optics , quantum mechanics , engineering
Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high‐pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild‐type GFP (wtGFP) and three of its red shift mutants (AFP, GFP mut1 , and GFP mut2 ) using Fourier transform infrared spectroscopy. For the wt GFP, GFP mut1 , and GFP mut2 we found that up to 13–14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3‐D structure is held responsible for this high‐pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure‐induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 244–253, 2002