Hyperfolder YFP

Excerpts

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.

Campbell et al. (2022)

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).

Campbell et al. (2022)