Benefiting from the hfYFP crystal structure, we eliminated hfYFP’s 514-nm excitability and produced two exclusively 405-nm excitable GFPs with [a large Stokes shift], LSSA12 and LSSmGFP. These LSS FPs overcome the cross-excitation problem of mT-Sapphire (Fig. 5c–e) without sacrificing molecular brightness (Table 1). LSSmGFP has high chemical and thermodynamic stability and the same molecular brightness as mAmetrine, while lasting twice as long under laser-scanning confocal illumination before photobleaching (Extended Data Fig. 7g).
Introducing the S147P mutation that was reported to improve thermostability in violet-light excitable uvGFP yielded hyperfolder mutants with considerable resistance to 1 M sodium hydroxide (NaOH) solutions of pH ≥ 13 (we were able to determine hyperfolder FP extinction coefficients by collecting alkaline denaturation time-course data) (Extended Data Fig. 5 and Supplementary Methods). hfYFP-S147P/V206K/L195M behaved as a stronger monomer in the OSER assay than did hfYFP (Extended Data Fig. 3d,e); the L195M mutation originated de novo from a PCR error. The V206K mutation (on β-strand 10) largely preserved the GdnHCl stability of multiple mutants (Extended Data Fig. 4d). We named the hfYFP-S147P/V206K/L195M variant ‘mhYFP’.
Consistent with earlier data [Campbell et al., PNAS, 2020], sfGFP, mClover3, mNeonGreen, and eYFP proteins fully denatured within 10 min after dilution into buffered 6.3 M guanidinium HCl (GdnHCl), whereas mGreenLantern (mGL) remained above its half-initial fluorescence value until ~200 min. Intriguingly, instead of dimming, hfYFP grew 50% brighter in 6.3 M GdnHCl. When measured after 12, 24 and 48 h, hfYFP fluorescence was unchanged relative to its value at 2 h (Fig. 2a). Whereas sfGFP denatured instantly in 7 M GdnHCl, hfYFP persisted >3 months in the same solution at room temperature (RT) (Fig. 2a, inset photograph). Similar to sfGFP, moxGFP denatured immediately upon exposure to GdnHCl, whereas hfYFP never denatured (Extended Data Fig. 4c). The melting temperature of hfYFP (Tm= 94.2 °C) was approximately 20 °C and 14 °C greater than eYFP’s and eGFP’s values, respectively. hfYFP and mGL can tolerate higher temperatures for much greater lengths of time than the other FPs, including sfGFP.
hfYFP tolerated deleterious mutations that rendered eGFP and even sfGFP almost entirely nonfunctional, suggesting that it will be a good template for mutagenesis and sensor engineering (Extended Data Fig. 2).
LSSA12 was more stable in guanidinium HCl (GdnHCl) than mT-Sapphire, mAmetrine, and eGFP (Extended Data Fig. 6e). Site-directed mutagenesis confirmed the functional importance of the E222D and G65S mutations in LSSA12 (Extended Data Fig. 6f). LSSmGFP and LSSA12 enjoy similar advantages as hfYFP, including the absence of cysteine residues, low pKa, tolerance of fixatives, high chemical and thermal stability, and a single excitation band.
In contrast to Clover (Extended Data Fig. 5a), sfGFP (Extended Data Fig. 5b), and all other avFPs we had tested, FOLD6 required 10 min to [denature] in 1 M NaOH solution (pH ≥ 13) (Extended Data Fig. 5c). We performed time-course experiments with eight different FPs and found that Clover, eYFP, and even mGL and hfYFP, denatured right away in 1 M NaOH (Extended Data Fig. 5d-g).