title: Temperature-pressure stability of green fluorescent protein: a Fourier transform infrared spectroscopy study.
abstract: 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.
authors.affiliation: Lehrstuhl für Technische Mikrobiologie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
authors.forename: Carsten H
authors.initials: CH
authors.lastname: Scheyhing
authors.forename: Filip
authors.initials: F
authors.lastname: Meersman
authors.forename: Matthias A
authors.initials: MA
authors.lastname: Ehrmann
authors.forename: Karel
authors.initials: K
authors.lastname: Heremans
authors.forename: Rudi F
authors.initials: RF
authors.lastname: Vogel
mesh_terms: D004355:Drug Stability; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011312:Pressure; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins