By observing the brightness of bacterial colonies expressing randomly mutated Dronpa genes, we identified Dronpa-V60A, a monomeric Dronpa mutant with increased in-colony brightness. In vitro maturation measurements showed that Dronpa-V60A forms almost three times faster than Dronpa, with a maturation half-time of less than 15 min. We thus called this mutant “fast-forming” or ffDronpa.
The red form of pcDronpa2 has the highest extinction coefficient of all PCFP red forms known to date and is, just like its ancestor pcDronpa, not photoswitchable in the red form. Rational mutagenesis based on literature examples (S142A, V157I/G/S, M159A/T, F173S/L)15,23,45,48 did not generate a four-way highlighter pcDronpa2 mutant (data not shown).
We found that reverting the N102I and E218G mutations is not a viable strategy to obtain a monomeric pcDronpa2 variant... As an alternative approach, we used our crystallographic data to rationally break the tetramer interfaces in a different way. Mutations N158E and Y188A disrupted the A/C interface, while V123T additionally broke the A/B interface. Although we could thus successfully make a monomeric version of pcDronpa2 (pcDronpa2-V123T-N158E-Y188A), this mutant displayed no photoconversion.