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.

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T

“…The protein is in the shape of a cylinder named β-can, consisting of 11 strands of β-sheet with an α-helix inside, which protect the fluorophore [49,50]. GFP is quite thermostable [51], very resistant to chemical and pressure-induced denaturation [52][53][54].…”

“…4) Pressure variations are immediately reported in the fluorescence spectrum. 5) The protein stability domain, which ranges up to at least 600 MPa [53,54], exceeds the pressure domain that is used in experiences performed with most live microorganisms or their cellular enzymatic machinery.…”

Insights into the role of hydration in protein structure and stability obtained through hydrostatic pressure studies

“…Often dictated by the technique used to observe the effects of pressure, changes in this parameter can lead to very different observations, since aggregation phenomena are favored at high concentrations. Thus, pressure-induced aggregation is often observed in high-pressure Fourier transform infrared studies in which high concentrations of protein are required (27)(28)(29)(30)(31)(32). In one case at high-Fourier transform infrared concentration, pressure led to the population of a partially unfolded dimer, whereas for the same system at the lower concentrations used in fluorescence, pressure led to total dissociation and unfolding (26).…”

Estudos de renaturação de proteínas agregadas utilizando altas pressões hidrostáticas

“…Foi demonstrado que as estruturas secundárias da GFP e também da eGFP, monitoradas por meio de estudos de espectroscopia de infravermelho (FT-IR), são mantidas em pressões de até 13 -14 kbar (Scheyhing, Meersman et al 2002), confirmando que as GFPs são altamente resistentes à desnaturação pela aplicação de pressão.…”

“…O espectro da eGFP nativa liofilizada na região de amida I (figura 16) mostra a presença de picos que podem ser atribuídos a elementos de estrutura secundária (Barth 2007 podem ser atribuídos a folhas-β com orientação antiparalela, o que está de acordo com os dados cristalográficos sobre a GFP nativa (Ormo, Cubitt et al 1996) e com o espectro de infravermelho da GFP selvagem (Scheyhing, Meersman et al 2002). A comparação entre os espectros de infravermelho dos CI produzidos em temperaturas variando entre 37ºC e 47ºC mostra que as alterações na temperatura de expressão da proteína induziram mudanças na região de amida I dos espectros.…”