Since the discovery of the Green Fluorescent Protein (GFP), fluorescent proteins (FPs) have been used as biomarkers to monitor biological phenomena across many scientific disciplines. All naturally-occurring FPs consist of an 11-stranded β-barrel with a non-canonical α-helix, which contains the chromophore, running through the central axis of the protein. Glycines 31, 33, and 35 are highly conserved across the fluorescent proteins found in the PDB. These three residues are of interest due to them all being located in the second strand of the β-barrel, but having no direct involvement in chromophore formation. This led to a presumption that the glycines likely allowed space in a correctly folded β-barrel for the chromophore to form.
In this study, molecular dynamics simulations of G31A, G33A, and G35A single point mutants of wild-type GFPs with immature (pre-cyclized) chromophores were used to investigate how mutations to these residue positions could affect chromophore formation. Four additional mutant simulations were performed to investigate the hydrophobic pocket that contains G35. This was done by examining the hydrogen bond network in the central α-helix, water migration through the β-barrel, aromatic rescue interactions, and main chain interactions among the N-terminus β-sheets. The simulations show that if the β-barrel folds correctly, mutating the conserved glycines does not result in hindrance or prevention of chromophore formation.
Through experimental analysis, it was found that the G3XA mutants were prone to misfold and aggregate, suggesting that these glycines play a crucial role in the folding pathway of fluorescent proteins. Computationally, this was confirmed as the introduced mutations caused reduced main chain interactions among the N-terminus β-sheets.
Nwafor, Justin, "Why are Glycines 31, 33, and 35 Highly Conserved in all Fluorescent Proteins?" (2021). Chemistry Honors Papers. 31.
The views expressed in this paper are solely those of the author.
Winner of 2021 Oakes Ames Prize for best senior honors paper.
This research was published in Biochemistry on 22 November 2021.