Denaturation studies reveal significant differences between GFP and blue fluorescent protein
Introduction
The green fluorescent protein (GFP) is an auto-fluorescent protein that was first identified and isolated from the jellyfish Aequorea victoria [1]. GFP is a compact protein of 238 amino acids consisting of a fluorophore composed of three modified amino acids (–Ser65–Tyr66–Gly67). Due to its auto-fluorescence both in vitro and in vivo, as well as its remarkable stability, it is widely used for numerous cell biology and molecular biology applications [1], [2]. Mutant forms of this protein that allow efficient in vivo folding, high levels of expression in E. coli, and increased and shifted fluorescence are increasingly used as fusion proteins for protein engineering, expression studies, as well as biotechnology applications [2], [3], [4], [5], [6], [7]. For example, Waldo et al. have reported on a technique to screen for properly folded recombinant proteins in E. coli, using a GFP fusion tag [8]. GFP variants have also been used in fluorescence resonance energy transfer assays to study protein–protein interactions in living cells [9]. Recently it has been shown that the GFP fluorescence is pH dependent, a property that has been exploited to use GFP as intracellular pH indicators [5], [10]. Additionally, we have shown that GFP could also be used to develop a sensitive and cheap assay to screen antioxidants [18]. One of the other interesting properties of GFP is its unusual stability to heat, pH, proteases, and denaturants, which is probably due to the tight and compact “β-can” structure of the GFP molecule [11], [12], [13], [14]. It is well documented and accepted that GFP fluorescence is intimately linked to its properly folded structure, as in the native structure the chromophore has restricted movement and is shielded from bulk water and only when the GFP is denatured, the chromophore has increased rotational freedom and also undergoes attack by water molecules leading to quenching of its fluorescence [11]. We and others have recently shown that GFP's unusual stability in various denaturants is pH dependent and could be studied by fluorescence spectroscopy [15], [16]. These observations led us to hypothesize that GFP probably undergoes a slight but significant structural shift between pH 6.5 and 7.5. This hypothesis is further supported by the crystallographic observations by Jain and Ranganathan that there are minor but significant structural changes in GFP between pH values of 8.5 and pH 5.5 [17]. Additionally, we have recently reported that oxyradicals can be used to denature GFP and that this oxyradical-dependent loss of GFP fluorescence is also pH dependent, such that GFP is most labile to oxy-radical induced damage at pH 6.5 as compared to pH 7.5 or pH 8.5 [18].
Although, a great deal of research has been published on the stability of GFP, similar studies on GFP's closely related variants like BFP, Yellow Fluorescent Protein (YFP), and Red Fluorescent Protein (RFP) are very scarce. Therefore, we wanted to examine whether the stability or structure of the closely related BFP is also pH dependent, as we and others have previously observed for GFP. During the course of our study, we found that denaturation of BFP, like that of the closely related GFP, was also pH dependent, however with significant and interesting differences. Additionally, it appears that BFP is more stable than GFP under certain denaturing conditions. These differences between GFP and BFP when exposed to different denaturants suggest that although both BFP and GFP share overall structures (β-barrel) and are almost identical in amino acid sequences, there maybe be subtle but significant differences between these two proteins. We feel that studies like this that explore unusual properties of these auto-fluorescent proteins will lead to their additional uses in novel biotechnology applications.
Section snippets
Materials
BFP (SuperGlo™ BFP) was purchased from Qbiogene (USA). Other reagents including buffers, SDS, and urea were purchased from Sigma–Aldrich (USA).
Cloning and purification of GFP
GFP was cloned into a pET vector after PCR amplifying the GFP gene from the pQBI T7-GFP plasmid (Qbiogene, USA). Details of cloning and purification has been described elsewhere [15], [19].
Fluorescence analysis
Fluorescence spectra of GFP and BFP were determined using the Cary Eclipse Fluorescence Spectrophotometer using a quartz fluorescence cell in 3 ml 50 mM Tris buffers.
Results and discussion
BFP was originally created by changing the amino acids in the vicinity of the chromophore of GFP [20]. As shown in Fig. 1, these two proteins are almost identical in amino acid sequence (more than 93% identity), with the major difference being in the chromophore region. As expected, the two proteins show complete alignment of their secondary structures (Fig. 2). In fact, even the X-ray crystal structures of the two proteins shows that the overall global structures are almost identical with an
Conclusion
In summary, our results show despite having very similar structures and being almost identical to GFP, BFP has significantly different physical properties and structural stability. Like the previously published data showing denaturation of GFP was pH dependent, BFP also showed different denaturation profiles at different pH values, however, with differences when compared to GFP. Also, it seems that under certain conditions (like pH 6.5), BFP appears to be more structurally stable and resistant
Acknowledgments
The authors thank the Research Affairs at the UAE University for funding this research (under a contract no. 01-03-2-11/07). We would also like to express our gratitude to undergraduate students who worked on this project—Mona Alnuaimi Maryam Nasser Shebli, and Amal Hekmani as well Dr. Mohammed Meetani, for his helpful insights.
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