Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: Insights for the development of pH-biosensors

Author(s)

dos Santos, Nathalia Vieira ^{[1, 2]} ; Saponi, Carolina Falaschi ^{[1, 2]} ; Ryan, Timothy M. ^[3] ; Primo, Fernando L. ^[2] ; Greaves, Tamar L. ^[1] ; Pereira, Jorge F. B. ^{[2, 4]}

Affiliation

^[1] RMIT Univ, Coll Sci Engn & Hlth, Sch Sci, 124 La Trobe St, Melbourne, Vic 3000 - Australia

^[2] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Engn Bioproc & Biotechnol, Rodovia Araraquara Jau Km 01, BR-14800903 Araraquara, SP - Brazil

^[3] Australian Synchrotron, 800 Blackburn Rd, Clayton, Vic 3168 - Australia

^[4] Univ Coimbra, Dept Chem Engn, CIEPQPF, Rua Silvio Lima, P-3030790 Coimbra - Portugal

• Document type: Journal article
• Source: International Journal of Biological Macromolecules; v. 164, p. 3474-3484, DEC 1 2020.
• Web of Science Citations: 0

Abstract

Enhanced Green Fluorescent Protein (EGFP) is a biomolecule with intense and natural fluorescence, with biological and medical applications. Although widely used as a biomarker in research, its application as a biosensor is limited by the lack of in-depth knowledge regarding its structure and behavior in adverse conditions. This study is focused on addressing this need by evaluating EGFP activity and structure at different pH using three-dimensional fluorescence, circular dichroism and small-angle X-ray scattering. The focus was on the reversibility of the process to gain insights for the development of biocompatible pH-biosensors. EGFP was highly stable at alkaline pH and quenched from neutral-to-acidic pH. Above pH 6.0, the fluorescence loss was almost completely reversible on return to neutral pH, but only partially reversible from pH 5.0 to 2.0. This work updates the knowledge regarding EGFP behavior in pH by accounting for the recent data on its structure. Hence, it is evident that EGFP presents the required properties for use as natural, biocompatible and environmentally friendly neutral to acidic pH-biosensors. (C) 2020 Elsevier B.V. All rights reserved. (AU)

• FAPESP's process: 18/20833-0 - Entendendo as interações moleculares e a estabilidade da proteína verde fluorescente (GFP) em líquidos iônicos sob condições de estresse
• Grantee: Carolina Falaschi Saponi
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• 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: 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: 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: 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)
• 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: 18/01858-2 - 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