Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP

Abstract

Enhanced green fluorescent protein (EGFP)—one of the most widely applied genetically encoded fluorescent probes—carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion (“redding”) with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants

“…While the separate effect of the G65 can be interpreted in terms of the increased oscillator strength of the G-Y-G chromophore [ 20 ], the combinations of T65G with both Y145M and F165Y probably strengthen this effect (giving an additional increase in EC from 70,000 in EGFP-T65G to ~85,000 M −1 cm −1 in T65G/Y145M, T65G/F165Y, and BrUSLEE). The introduction of F165Y into EGFP-T65G resulted in a sufficient FQY improvement, thus providing evidence of the possible role of Y165 in an excited-state chromophore stabilization (similar to its functioning in BrUSLEE [ 12 ]).…”

“…In the parental EGFP [ 16 ], the FL value was reported to range from 2.3 to 3.4 ns [ 9 , 17 , 18 , 19 ], depending on the excitation regime and external conditions. The T65G mutation of the chromophore-forming residue drastically reduces EGFP brightness and shortens the lifetime to 1.3 ns [ 12 , 17 , 20 ]. We thus consider this substitution as the primary and key gateway to the FL change in the BrUSLEE relative to EGFP, probably due to a decrease in the average number of hydrogen bonds between the chromophore and its environment, resulting in an increase in chromophore flexibility and changes in its excited-state dynamics [ 20 ].…”

“…The T65G mutation of the chromophore-forming residue drastically reduces EGFP brightness and shortens the lifetime to 1.3 ns [ 12 , 17 , 20 ]. We thus consider this substitution as the primary and key gateway to the FL change in the BrUSLEE relative to EGFP, probably due to a decrease in the average number of hydrogen bonds between the chromophore and its environment, resulting in an increase in chromophore flexibility and changes in its excited-state dynamics [ 20 ]. However, FL and brightness are both established through the cooperative action of the chromophore and chromophore-surrounding residues.…”

“…However, FL and brightness are both established through the cooperative action of the chromophore and chromophore-surrounding residues. For instance, in the enhanced yellow fluorescent protein (EYFP), the G65/Y203 combination provides a FL of 3–3.2 ns and a FQY of 0.61 [ 20 ]. Introduction of the second mutation, Y145M, leads to further FL reduction down to 800 ps in the T65G/Y145M mutant [ 12 ].…”

Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics

“…Occasionally, more conventional fluorophores such as GFP and its variants, eGFP and YFP, may exhibit fluorescence intensity fluctuations under special conditions of excitation at or out of their nominal excitation wavelength (reviewed in Bagshaw and Cherny, 2006). For example, eGFP was shown to blink at acidic pH values when illuminated with a 405 nm laser (Haupts et al, 1998) or it may exhibit oxidative photoconversion to a red emitter following excitation with a 532 nm laser line (Sen et al, 2019). The versatility of more conventional fluorophores in SMLM has led to the development of unique methods including Stochastic Optical Fluctuations Imaging (SOFI; Dertinger et al, 2009) or Bayesian analysis of blinking and bleaching (3B; Cox et al, 2012) with a very big potential to broaden SMLM applications in plants.…”