Fluorescent imaging is widely used for <i>in vivo</i> tracking, quantification and visualization of cellular processes, many of which are pH-dependent. This drives the need to develop non-invasive pH-sensors, such as those based on genetically encodable fluorescent proteins (FP). Here, we used a combination of the weighted ensemble sampling method  (WE), constant pH molecular dynamics simulations (CPHMD) and quantum–mechanical calculations to elucidate the mechanism of unusual pH-dependent spectral behavior of a recently engineered mutant of cyan fluorescent protein (WasCFP). This mutant exhibits a pH-dependent absorption suggesting that its tryptophan-based chromophore switches between cyan-emitting neutral (protonated) and green-emitting charged (deprotonated) states depending on external pH. Such an unusual behavior opens new avenues for the development of pH sensors, but its mechanistic understanding remains elusive. We discovered the existence of partially solvated open state that is transiently populated at low pH but becomes dominant under mildly basic conditions, and, thus, accounts for the pH-mediated transition from cyan to green fluorescent forms of WasCFP. To bolster a mechanistic understanding of WasCFP, we developed a three-state theoretical model that fully explains its unusual pH-dependent spectral properties, which suggests that tuning the thermodynamic stability of various conformations can be used a novel approach for engineering new FP variants.<br/> <br/>

Fluorescent proteins as pH sensors: Insights from constant pH molecular dynamics

249th National Meeting (2015)

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