Short communicationThermal stability of chemically denatured green fluorescent protein (GFP): A preliminary study
Introduction
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is one of the most widely studied and applied proteins in biochemistry and cell biology. GFP converts the blue light (that would otherwise be emitted by the Ca2+-sensitive protein aequorin) into a brilliant green fluorescence [1], [2], [3]. GFP and its genetically modified variants are widely used as fluorescent biosensors for protein expression and to study the dynamics and protein–protein interactions in living cells [4], [5].
GFP (28 kDa, 238-aa residues) is a barrel-shaped molecule, 24 Å in diameter and 42 Å in length. Outer “layer” of the barrel is composed of 11 antiparalell β sheets. Antiparalell sheets are connected with α-helical stretches. One α-helix extends to the interior of the “β-can” and forms the fluorescent chromophore. Chromophore is composed from three (–Ser65–Tyr66–Gly67) post-translationally modified amino acids [6]. Fluorescent properties of GFP and its genetically modified variants are determined by interaction between these three amino acids and neighboring residues. Extensive digestion of GFP with papain has yielded a hexapeptide (Phe64–Gln69), which contains tripeptide, but this fragment has been found nonfluorescent [7].
Enhanced GFP (EGFP) is a mutant of GFP with 35-fold increase in fluorescence [8], [9], [10] This variant has mutations of Ser to Thr at amino acid 65 and Phe to Leu at positions 64 and encoded by gene with optimized human codons [9].
Thermally denatured GFP can be renatured at the low temperature, so the process is reversible [11], [12]. Denaturating agents, such as GuHCl lowers the temperature of denaturation of GFP, which can be monitored by differential scanning calorimetry. Thermally or chemically unfolded polipeptide cannot be an ideal mathematically random chain. There is considerable experimental evidence for local order in proteins denatured by different denaturants [13], [14], [15]. It is interesting to check the simultaneous influence of the two denaturing processes and the question of reversibility of the unfolding for GFP because intrinsic viscosity data given at identical temperatures suggest that unfolded chains evoked either heat treatment or GuHCl are very nearly random coils. The free energy associated with both kind of denaturation are very similar at the same temperature and pH [16]. Therefore, in this study we present data for the effect of denaturing agent (GuHCl) on the global thermal stability of GFP.
Section snippets
Cloning of EGFP
E-GFP clone was purchased from BD Biosciences, Clontech. pEGFP-N1 encodes a red-shifted variant of wild-type GFP [17], [18], [19] which has been optimized for brighter fluorescence and higher expression in mammalian cells (excitation maximum=488 nm; emission maximum=507 nm). pEGFP-N1 encodes the GFPmut1 variant [6] which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond
Results and discussion
Since each amino acid influences the free enthalpy of both the folded and unfolded states, insight into denaturated proteins is crucial for understanding protein stability [20]. During unfolding the polypeptide chain becomes less compact, more highly solvated and much more flexible [21]. According to our measurements the native GFP seems to be very heat stable. It has one endotherm with 83 °C melting temperature and 56 J/kg calorimetric enthalpy in 0–100 °C temperature range (Fig. 1). Its thermal
Acknowledgements
This work was supported by grants of OTKA CO-272 (D.L.).
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