Interplay of Radiative and Non Radiative Rate Constants in the Photophysics of Fluorescent Proteins

Abstract

Genetically encodable fluorophores like Fluorescent Proteins (FPs) have been engineered to provide better contrast, localization and specificity. Red-FPs (RFPs) are known to have an extended chromophore through an acylimine moiety with respect to their blue shifted and generally brighter counterparts. The molecular brightness is based on the product of the extinction coefficient (EC) and fluorescence quantum yield (QY). Development of brighter FPs has focused on increasing the QY, which is directly proportional to fluorescence lifetime (FL), a time constant corresponding to the inverse sum of the ensemble radiative and non-radiative decay rate constants. We have performed directed evolution of RFPs through random and site-directed mutagenesis on a multi-parametric microfluidic sorting system capable of FL and brightness selections. We also applied site directed mutagenesis on RFPs based on structural analysis to investigate the structural effects on FL and QY. The selected mutants derived from mCherry and FusionRed show that the improvement in FL brings about an expected linear increase in QY (∼3.2 fold for mCherry and ∼2.3 fold for FusionRed). This can be attributed to significant decrease in the non-radiative rate constant, and a modest improvement in radiative rate constant that is supported by hypsochromic shifts and changes in absorption profiles. Both approaches reveal that the improvement in molecular brightness with increased FL is mainly governed by a decrease in the non-radiative rate constant in conjunction with improved absorption (narrower absorption spectrum and higher ECmax). This strategy forms the basis of the best photophysical route to engineer brighter RFPs.