Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu

Abstract

The autocatalytic maturation of the chromophore in green fluorescent protein (GFP) was thought to require the precise positioning of the side chains surrounding it in the core of the protein, many of which are strongly conserved among homologous fluorescent proteins. In this study, we screened for green fluorescence in an exhaustive set of point mutations of seven residues that make up the chromophore microenvironment, excluding R96 and E222 because mutations at these positions have been previously characterized. Contrary to expectations, nearly all amino acids were tolerated at all seven positions. Only four point mutations knocked out fluorescence entirely. However, chromophore maturation was found to be slower and/or fluorescence reduced in several cases. Selected combinations of mutations showed nonadditive effects, including cooperativity and rescue. The results provide guidelines for the computational engineering of GFPs.

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

“…The F39G mutation blocked formation of the anionic form of the FGCaMP indicator ( Figure S2b,c). F39 (F165 in GFP) seemed to create a non-polar environment around the chromophore and hold the polar core (composed of R215 and Q213 residues) around the chromophore ( Figure S2d), as has been earlier predicted for GFP [30]. Furthermore, the F39 residue may provide hydrophobicity and participate in stacking interactions with the chromophore, thus stabilizing the anionic form of FGCaMP.…”

“…The Q213A mutation (position Q94 in GFP) slightly increased the Abs 402 /Abs 493 ratio, probably due to the removal of hydrogen bond (H-bond) donors/acceptors near the imidazolinone ring of the chromophore ( Figure S2c,d). Therefore, the H-bond donor/acceptors in position 213 and positive charge in position R215 are important for stabilization of the FGCaMP anionic form in Ca 2+ -saturated state, due to the support of the polar chromophore environment; similar to that in GFP [30]. T181 (T62 in GFP) and Q213 residues in FGCaMP create a polar environment ( Figure S2d), stabilizing the anionic 493 nm-absorbing form of FGCaMP, as has been shown earlier for GFP [30,31].…”

“…Therefore, the H-bond donor/acceptors in position 213 and positive charge in position R215 are important for stabilization of the FGCaMP anionic form in Ca 2+ -saturated state, due to the support of the polar chromophore environment; similar to that in GFP [30]. T181 (T62 in GFP) and Q213 residues in FGCaMP create a polar environment ( Figure S2d), stabilizing the anionic 493 nm-absorbing form of FGCaMP, as has been shown earlier for GFP [30,31]. Probably for this reason, the T181G mutation lacked a 493 nm absorbing form and showed only a protonated 402 nm absorbing form ( Figure S2b).…”

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process

“…Quantum yields for the complemented sGFP variants display a similar trend but are generally 10% lower than those of flGFPs, except for sGFP3, which suffered a large loss of quantum yield (Φ = 0.29) (Table 1 ). Such reduced quantum yields are consistent with the decreased fluorescence lifetimes observed for complemented sGFP variants compared to flGFPs and suggest that the β-barrel surrounding the chromophore in re-assembled sFPs is less rigid than in flFPs, allowing the chromophore to be relatively mobile 47 , 48 .…”

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design

“…Here we show that a single insertion of the different substituents at the EYFP 165th position can give a variety of outcomes – from weakly UV-fluorescent protein with severely impaired maturation (165G, 165S) to extremely bright variants (165H or 165H/148S) that outperform the original EYFP, show similar characteristics to LanYFPs and almost twofold higher brightness than such yellow-emitting probes as phiYFP, mCitrine, YPet [34] . Although phenylalanine-165 was previously targeted for mutagenesis aimed at the parental protein fluorescence alteration (for example, when creating a FLIM-FRET acceptor REACh [48] , and even studied systematically by the saturation mutagenesis in OPT-GFP, a close EYFP homologue [49] , 165th position mutations have never been described as dramatically affecting the protein characteristics. Thus, in the latter case, F165 position was described as tolerating all mutations.…”