Reverse pH-dependence of chromophore protonation explains the large Stokes shift of the red fluorescent protein mKeima.J Am Chem Soc 2009; 131(30):10356-7JA
The recently developed red fluorescent protein Keima exhibits the largest Stokes shift (180 nm) observed to date. Combining X-ray crystallography with (in crystallo) UV-visible absorption, fluorescence, and Raman spectroscopy, we have investigated molecular determinants of this peculiar property. The results demonstrate a pH-dependent "reverse chromophore protonation" triggered by the key residue Asp157 and which couples to cis/trans isomerization of the chromophore. These data provided guidelines to rationally design a useful Keima variant.