Decoding mEos4b day‐long maturation and engineering fast‐maturing variants

Maity A, Glushonkov O, Ayala I, Tacnet P, Wulffelé J, Frachet P, Brutscher B, Bourgeois D, Adam V

(2025). Protein Science, 34(8) , e70234. doi: 10.1002/pro.70234. Article   Pubmed

    Primary Proteins:
  1. mEos4b-L93M
  2. mEos4Fast1
  3. mEos4Fast2
    Secondary Proteins:
  1. mEos3.1,
  2. mEos3.2,
  3. mEos4b,
  4. mEosEM,
  5. pcStar
Add photostability measurements

Excerpts

Our measurements showed that, while the mutation V69A further slowed down the maturation kinetics of mEos4b, the three other single-point mutants improved it. Despite this, the V69T and I157V mutations were still unsatisfactory, with maturation time constants exceeding 400 min. In contrast, the L93M mutant showed a drastically decreased apparent maturation time of approximately 80 min, a more than 20-fold improvement relative to the mEos4b parent and better than anyother L93 substitution we have tested.

In contrast, the T59Q mutation significantly improved the apparent maturation time, particularly in the variant mEos4b-T59Q-L93M, which we renamed mEos4Fast2. This protein outperforms any other tested mEos variants and challenges the very fast-maturing mEosEM and pcStar.

Introducing the single A60Q mutation resulted in maturation times of 209 ± 13 min for mEos4b-A60Q and 64 ± 5 min for mEos4b-A60Q-L93M. We named this fast-maturing double mutant mEos4Fast1.