Kinetic isotope effect reveals rate‐limiting step in green‐to‐red photoconvertible fluorescent proteins

Abstract

AbstractPhotoconvertible fluorescent proteins (pcFPs) undergo a slow photochemical transformation when irradiated with blue light. Since their emission is shifted from green to red, pcFPs serve as convenient fusion tags in several cutting‐edge biological imaging technologies. Here, a pcFP termed the Least Evolved Ancestor (LEA) was used as a model system to determine the rate‐limiting step of photoconversion. Perdeuterated histidine residues were introduced by isotopic enrichment and chromophore content was monitored by absorbance. pH‐dependent photoconversion experiments were carried out by exposure to 405‐nm light followed by dark equilibration. The loss of green chromophore correlated well with the rise of red, and maximum photoconversion rates were observed at pH 6.5 (0.059 ± 0.001 min−1 for red color acquisition). The loss of green and the rise of red provided deuterium kinetic isotope effects (DKIEs) that were identical within error, 2.9 ± 0.9 and 3.8 ± 0.6, respectively. These data indicate that there is one rate‐determining step in the light reactions of photoconversion, and that CH bond cleavage occurs in the transition state of this step. We propose that these reactions are rate‐limited on the min time scale by the abstraction of a proton at the His62 beta‐carbon. A conformational intermediate such as a twisted or isomerized chromophore is proposed to slowly equilibrate in the dark to generate the red form. Additionally, His62 may shuttle protons to activate Glu211 to serve as a general base, while also facilitating beta‐elimination. This idea is supported by a recent X‐ray structure of methylated His62.