Investigating an Unusually Bright Variant of the Red Fluorescent Protein mKeima

Presenter: A.J. Risenmay, Biology

mKeima is a monomeric red fluorescent protein (λem¬¬max ~620 nm) that is maximally excited in the blue (λex¬¬max ~440 nm). This extraordinarily large stokes shift can be significantly reduced following chromophore deprotonation under acidic conditions. By designing mutants to exploit the varying excitation species of mKeima, our lab was able to develop a redox-sensitive red fluorescent protein to be used as a quantitative reporter of the thiol/disulfide status in reducing subcellular compartments. This ratiometric variant was identified as mKeima M159K_TDCC and was found to be unusually fluorescent under green light (580 nm). Further examination revealed that mKeima M159K_TDCC is colorless when expressed in the dark, but irreversibly becomes pink when exposed to blue light. Here we combine x-ray crystallography and fluorescence/absorption spectroscopic techniques to investigate the fascinating chemistry behind this mKeima mutant.