Denaturation studies reveal significant differences between GFP and blue fluorescent protein

https://doi.org/10.1016/j.ijbiomac.2009.05.010Get rights and content

Abstract

Green fluorescent protein (GFP) is an unusually stable fluorescent protein that belongs to a family of related auto-fluorescent proteins (AFPs). These AFPs have been generated from jellyfish GFP by mutating the amino acids in the chromophore or its vicinity. Variants that emit light in the blue region (Blue Fluorescent Protein, BFP), red region, or yellow region are readily available and are widely used in diverse applications. Previously, we had used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with SDS, urea, and heat. Surprisingly, we found that SDS, urea or heat, did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5, however, at pH 6.5, the protein lost all fluorescence within a very short period of time. These results suggested that GFP undergoes a structural/stability shift between pH 6.5 and 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat. In the present study, we wanted to explore whether the stability or structure of the closely related BFP is also pH dependent. As expected, we found heat-induced denaturation and renaturation of BFP to be pH dependent, very much like GFP. However, when exposed to other denaturants like urea/heat or SDS we found BFP to behave very differently than GFP. Unlike GFP, which at pH 8.5 and 7.5 is very resistant to SDS-induced denaturation, BFP readily lost about 20% of its fluorescence at pH 8.5 and about 60% fluorescence at pH 7.5. Also, our denaturation and renaturation studies show that under certain conditions, BFP is more stable than GFP, such that under conditions where GFP is completely denatured, BFP still retained significant fluorescence. Taken together, our preliminary results show that despite being very similar in both amino acid sequences and overall structures, there may be subtle and important structural/conformational differences between BFP and GFP.

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.

References (22)

  • B. Philipps et al.

    J. Mol. Biol.

    (2003)
  • T. Zal et al.

    Cur. Opin. Immunol.

    (2004)
  • M. Kneen et al.

    Biophys. J.

    (1998)
  • J.R. Huang et al.

    J. Mol. Biol.

    (2007)
  • T. Aoki et al.

    Anal. Biochem.

    (2003)
  • A.A. Alnuami et al.

    Int. J. Biol. Macromol.

    (2008)
  • A. Nagy et al.

    Thermochim. Acta

    (2004)
  • R.Y. Tsien

    Annu. Rev. Biochem.

    (1998)
  • J.C. March et al.

    Appl. Microbiol. Biotechnol.

    (2003)
  • A. Muller-Taubenberger et al.

    Appl. Microbiol. Biotechnol.

    (2007)
  • N.C. Shaner et al.

    J. Cell Sci.

    (2007)
  • Cited by (30)

    • Imidazolium-based ionic liquids as additives to preserve the Enhanced Green Fluorescent Protein fluorescent activity

      2021, Green Chemical Engineering
      Citation Excerpt :

      The lower pH of the GuHCl 4 mol L−1 (6.09) and H2O2 1.50 mol L−1 (6.70) (Table S2 from the SI) may explain the lower protective aptitudes of the ILs for these conditions, considering EGFP changes states (from deprotonated to protonated) between pH 7 and 6 [2]. The protonated state of EGFP variants is more sensitive to denaturation (e.g., heat, urea, SDS) [2,18,19] and could explain why the [Cnmim]Cl ILs were less effective in protecting EGFP in more acidic conditions. Regarding the mechanisms involved for the protective effect of [Cnmim]Cl solutions on EGFP fluorescence against denaturing agents, it would be complex to isolate the variables responsible for this phenomenon, considering these protective effects could be not only due to specific interactions between the protein and ILs, but also from specific interactions between denaturing agents and ILs, or even a combination of different mechanisms from all the compounds in solution.

    • A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening

      2018, Analytical Biochemistry
      Citation Excerpt :

      Moreover, for a simple and fast demonstration of cleavage of the desired substrate with a specific enzyme, the fluorescent substrates and the generated products can be visualized by gel electrophoresis (Fig. 3). Our observations were in agreement with the previous observations of Saeed and its co-workers, who found that the fluorescent proteins exposed to denaturants show differences in their stability despite the close relationships [35]. Therefore, abilities of proteins for renaturation need to be considered in the experimental design, fluorescent proteins with higher stability need to be chosen or in-gel renaturation needs to be further optimized to make the detection of less-stable proteins sensitive enough.

    • Engineering and purification of a thermostable, high-yield, variant of PfCRT, the Plasmodium falciparum chloroquine resistance transporter

      2018, Protein Expression and Purification
      Citation Excerpt :

      Additionally, there has been significant success using C-terminally appended GFP tags to indicate if a membrane protein has been correctly processed and inserted into the plasma membrane of yeast, or prokaryotic, cells [33]. It also has been shown that GFP is SDS resistant and remains folded when analysed by SDS-PAGE [34]. Although there is much academic interest in PfCRT, there is very little literature precedence for its purification.

    • Meltable magnetic biocomposites for controlled release

      2017, Journal of Magnetism and Magnetic Materials
      Citation Excerpt :

      It was found that the fluorescence intensity decreased only slightly to 92% after 6 heating cycles. This result corresponds with published data [14]. The temperatures of the samples were determined with a fiber optical sensor (OPTOcon, Dresden, Germany).

    • Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B)

      2015, Biochimica et Biophysica Acta - Biomembranes
      Citation Excerpt :

      This was important because a significant reduction in the expression level of a TetA(B) mutant would give the same loss of tetracycline resistance phenotype to E. coli as a mutation that prevents conformation changes in the transporter. GFP remains fluorescent even in the presence of SDS [53], therefore SDS-PAGE was used to measure the expression levels of each mutant by in-gel fluorescence (Fig. 3). This was found to be more reliable than measuring whole-cell fluorescence.

    • Borrowing a little from research to enhance undergraduate teaching

      2010, Procedia - Social and Behavioral Sciences
    View all citing articles on Scopus
    View full text