Creating Light from Darkness- Engineering the GFP Fluorophore into a Colourless Protein
Start Date
April 2025
Location
3rd floor - Library
Abstract
Green fluorescent protein (GFP) exhibits fluorescence due to a fluorophore formed by the cyclization of three specific residues (Ser65, Tyr66, and Gly67) located within an alpha helix and enclosed by a protective beta-barrel structure. The aim of this study was to identify a non-fluorescent protein in the Protein Data Bank (PDB) that contained a similar three-residue motif, with the goal of engineering it to fluoresce in a manner similar to GFP. Building on previous work by Jessica Ziegler, the protein 1INL was identified as a promising candidate. 1INL is a spermidine synthase from Thermotoga maritima that forms a tetramer, with each monomer composed of multiple alpha helices. Structural analysis of 1INL suggests that its inability to form a fluorescent chromophore arises from the absence of stabilizing residues around the key three-residue motif, preventing cyclization. Chimera X was used to identify residues in 1INL that could be mutated into the specific supporting residues that facilitate the autocyclization of the fluorophore. We then used AlphaFold to predict how the mutated strain of 1INL would fold in 3D to ensure the structure of the protein was not disrupted. A gene for expression of the mutated protein in E. coli will be ordered from a company, and the ability for the protein to form the fluorophore will be evaluated.
Creating Light from Darkness- Engineering the GFP Fluorophore into a Colourless Protein
3rd floor - Library
Green fluorescent protein (GFP) exhibits fluorescence due to a fluorophore formed by the cyclization of three specific residues (Ser65, Tyr66, and Gly67) located within an alpha helix and enclosed by a protective beta-barrel structure. The aim of this study was to identify a non-fluorescent protein in the Protein Data Bank (PDB) that contained a similar three-residue motif, with the goal of engineering it to fluoresce in a manner similar to GFP. Building on previous work by Jessica Ziegler, the protein 1INL was identified as a promising candidate. 1INL is a spermidine synthase from Thermotoga maritima that forms a tetramer, with each monomer composed of multiple alpha helices. Structural analysis of 1INL suggests that its inability to form a fluorescent chromophore arises from the absence of stabilizing residues around the key three-residue motif, preventing cyclization. Chimera X was used to identify residues in 1INL that could be mutated into the specific supporting residues that facilitate the autocyclization of the fluorophore. We then used AlphaFold to predict how the mutated strain of 1INL would fold in 3D to ensure the structure of the protein was not disrupted. A gene for expression of the mutated protein in E. coli will be ordered from a company, and the ability for the protein to form the fluorophore will be evaluated.
