Spectral properties of WasCFP and their explanation.
(a) WasCFP absorption spectra at various pHs (solid colored lines) at 4°C with conversion of cyan to green-emitting forms. Dashed green line – emission at pH 8.1. Gray line – absorption of mCerulean denaturated in strong alkaline conditions. (b) Temperature dependence of WasCFP absorption spectra (at pH7.4). (c) A proposed explanation of the bathochromic shift in WasCFP. Neutral cyan chromophore (left) undergoes ionization upon pH increase. Resulting green-emitting anionic state of the chromophore (right) is stabilized by positively charged lysine-61 side chain. Photo inset shows fluorescence of WasCFP solution under UV lamp at pH 5 (left) and pH 8 (right).