Green Fluorescent Protein (GFP) has grown in popularity and new applications are currently being developed. There are certain residues that are highly conserved through all the naturally occurring fluorescent proteins variants, and some of their functionality is yet to be determined. This is the case for three glycines that appear in a GXGXGX motif in the second β-sheet at positions 31, 33, and 35 with conservations of 100%, 87% and 95%, respectively.
Molecular dynamic simulations and other computational analyses of G31A, G33A, and G35A mutants, derived from pre-cyclized wild-type GFP, determined with confidence that these glycines are not involved in the chromophore formation. It is now suspected that these glycines contribute to the folding pathways of the β-barrel, due to their innate flexibility and small size. Key distances within the structures were measured such as the hydrogen bond network of the ɑ-helix and tight turn to possibly determine the glycines functionality. Quantification of water channels within the protein was completed for all the mutants in order to determine the water migration pathways within the β-barrel. It was determined that the number of overall channels increased, but those with the directionality towards the ɑ-helical region decreased.
Other mutants of the precyclized wild-type GFP included G35V, F71L, F71Y, and G35V/F71L. These mutations were aimed to explore the steric effects and aromatic rescue interactions between the glycines and their neighbouring strands (β1-3). It was determined that there was an increase in the distances within the H-bond network of these mutants, decreasing the rigidity of the β-barrel. The biggest increase was seen in G35V/F71L, due to steric effects, and F71Y, possibly due to steric effects and the charge character that was added.
Salguero, Christian, "Why are Gly31, Gly33 and Gly35 highly conserved in all fluorescent proteins?" (2021). Chemistry Honors Papers. 30.
The views expressed in this paper are solely those of the author.