Why Are Gly31, Gly33, and Gly35 Highly Conserved in All Fluorescent Proteins?

• Published in: Biochemistry (Easton) Vol. 60; no. 49; pp. 3762 - 3770
• Main Authors: Nwafor, Justin, Salguero, Christian, Welcome, Franceine, Durmus, Sercan, Glasser, Rachel N, Zimmer, Marc, Schneider, Tanya L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14.12.2021
• Subjects: Alanine - chemistry; Alanine - metabolism; Amino Acid Motifs; Amino Acid Substitution; Cloning, Molecular; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Glycine - chemistry; Glycine - metabolism; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Molecular Dynamics Simulation; Mutation; Protein Conformation, beta-Strand; Protein Folding; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Structure-Activity Relationship; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.1c00587

Green fluorescent protein (GFP)-like fluorescent proteins have been found in more than 120 species. Although the proteins have little sequence identity, Gly31, 33, and 35 are 87, 100, and 95% conserved across all species, respectively. All GFP-like proteins have a β-barrel structure composed of 11 β-sheets, and the 3 conserved glycines are located in the second β-sheet. Molecular dynamics (MD) simulations have shown that mutating one or more of the glycines to alanines most likely does not reduce chromophore formation in correctly folded immature fluorescent proteins. MD and protein characterization of alanine mutants indicate that mutation of the conserved glycines leads to misfolding. Gly31, 33, and 35 are essential to maintain the integrity of the β1–3 triad that is the last structural element to slot in place in the formation of the canonical fluorescent protein β-barrel. Glycines located in β-sheets may have a similar role in the formation of other non-GFP β-barrels.