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Denaturation studies reveal significant differences between GFP and blue fluorescent protein.
Int J Biol Macromol. 2009 Oct 01; 45(3):236-41.IJ

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.

Authors+Show Affiliations

Department of Chemistry, UAE University, Al-Ain, United Arab Emirates.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19501614

Citation

Saeed, Ibtesam A., and S Salman Ashraf. "Denaturation Studies Reveal Significant Differences Between GFP and Blue Fluorescent Protein." International Journal of Biological Macromolecules, vol. 45, no. 3, 2009, pp. 236-41.
Saeed IA, Ashraf SS. Denaturation studies reveal significant differences between GFP and blue fluorescent protein. Int J Biol Macromol. 2009;45(3):236-41.
Saeed, I. A., & Ashraf, S. S. (2009). Denaturation studies reveal significant differences between GFP and blue fluorescent protein. International Journal of Biological Macromolecules, 45(3), 236-41. https://doi.org/10.1016/j.ijbiomac.2009.05.010
Saeed IA, Ashraf SS. Denaturation Studies Reveal Significant Differences Between GFP and Blue Fluorescent Protein. Int J Biol Macromol. 2009 Oct 1;45(3):236-41. PubMed PMID: 19501614.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Denaturation studies reveal significant differences between GFP and blue fluorescent protein. AU - Saeed,Ibtesam A, AU - Ashraf,S Salman, Y1 - 2009/06/06/ PY - 2009/02/01/received PY - 2009/05/19/revised PY - 2009/05/20/accepted PY - 2009/6/9/entrez PY - 2009/6/9/pubmed PY - 2009/10/20/medline SP - 236 EP - 41 JF - International journal of biological macromolecules JO - Int. J. Biol. Macromol. VL - 45 IS - 3 N2 - 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. SN - 1879-0003 UR - https://www.unboundmedicine.com/medline/citation/19501614/Denaturation_studies_reveal_significant_differences_between_GFP_and_blue_fluorescent_protein_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0141-8130(09)00120-2 DB - PRIME DP - Unbound Medicine ER -