Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy

Author(s)

dos Santos, Nathalia Vieira ^{[1, 2]} ; Saponi, Carolina Falaschi ^{[1, 2]} ; Greaves, Tamar Louise ^[2] ; Brandao Pereira, Jorge Fernando ^[1]

Affiliation

^[1] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Bioproc & Biotechnol, Rodovia Araraquara Jau Km 01, BR-14800903 Araraquara, SP - Brazil

^[2] RMIT Univ, Coll Sci Engn & Hlth, Sch Sci, 124 La Trobe St, Melbourne, Vic 3000 - Australia

• Document type: Journal article
• Source: RSC ADVANCES; v. 9, n. 40, p. 22853-22858, AUG 1 2019.
• Web of Science Citations: 0

Abstract

Fluorescent proteins have many applications as biomarkers and biosensors in the medical and biological fields. Their success was largely supported by modifications of the first isolated fluorescent protein, the wild-type Green Fluorescent Protein (wtGFP), which allowed the development of improved variants such as the Enhanced GFP (EGFP). The first reports on EGFP indicated that the protein presented a single form and fluorescence peak, in contrast to the two conformations observed in wtGFP. However, after experimental determination of the crystalline structure of EGFP, two conformations were found, generating questions regarding the relationship between EGFP structure and its spectral characteristics. To resolve the controversy, this study evaluated EGFP 3D fluorescence spectra at lower wavelengths and under distinct conditions (different concentrations, pH and temperatures), revealing the existence of a second fluorescence peak for this protein. It was possible to confirm that the new peak was not a reflection of the intrinsic fluorescence of proteins or an artefact from the 3D fluorescence spectroscopy. It was also shown that the second peak is pH dependent, sensitive to high temperatures and linearly related to EGFP concentration, confirming a direct relationship between the new fluorescence peak and EGFP protein structure. In addition to the revelation of the new EGFP fluorescence peak, this study demonstrated that 3D fluorescence can be used as powerful technique in the discovery of other elusive fluorophores. (AU)

• FAPESP's process: 14/16424-7 - Optimization and scale-up of liquid-liquid extraction process with ionic liquids (ILs) as a sustainable tool for the separation of the anti-leukemia biopharmaceutical L-asparaginase (ASPase)
• Grantee: Jorge Pereira
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 18/50009-8 - Understanding the molecular interactions between ionic liquids and biopharmaceuticals: the key for a proper design of downstream processing
• Grantee: Jorge Pereira
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/06576-5 - Molecular interactions and stability of Green Fluorescent Protein (GFP) in ionic liquids and encapsulation of GFP on reverse micelles
• Grantee: Nathalia Vieira Porphirio Veríssimo
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• FAPESP's process: 18/01858-2 - Study of the stabilizing effect of different solvents on the green fluorescent protein structure produced by Escherichia coli
• Grantee: Carolina Falaschi Saponi
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 14/19793-3 - Optimization and scale-up of novel Ionic-Liquid-based purification processes for recombinant green fluorescent protein produced by Escherichia coli "GFPurIL"
• Grantee: Sandro Roberto Valentini
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/07529-5 - Development of liquid-liquid purification process and incorporation in nanostructured polymeric matrix of recombinant green fluorescent protein produced by Escherichia coli
• Grantee: Nathalia Vieira Porphirio Veríssimo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)