A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore

Abstract

The far-red fluorescent protein mKate ( ex , 588 nm; em , 635 nm; chromophore-forming triad Met 63 -Tyr 64 -Gly 65 ), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 Å . The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. Green fluorescent proteins (GFP)2 and GFP-like proteins (FP) have become important noninvasive tools for visualization and monitoring of the internal processes within cells or whole organisms, such as gene expression, monitoring the cellular pH, ion concentration, embryogenesis, inflammatory processes, tracking protein trafficking, the migration of parasites within a host, etc (1-13). Fluorescent proteins can be used to visualize many types of cancer processes, including primary tumor growth, tumor cell motility and invasion, metastatic seeding and colonization, angiogenesis, and interactions between the tumor and its host microenvironment (14 -16). FPs might be very useful in real-time testing of the efficacy of cancer drugs in animal models of human cancer.The extensive spectral diversity of fluorescent proteins arises mostly from variations in the chemical structure of the mature chromophore and in the stereochemistry of its adjacent environment. The FP chromophore forms autocatalytically in vivo and in vitro from three residues, Xxx-Tyr-Gly, without need for any cofactors or enzymes, except for molecular oxygen (17). In most cases, the post-translational modification results in a blue/green emitting state, characterized by formation of an imidazolinone heterocycle with a p-hydroxybenzylidene substituent. Often, the reaction chain propagates further with formation of an additional N-acylimine double bond, which extends the conjugation of the chromophore electronic system and results in a bathochromic shift in spectra (18 -22).Proteins that emit red, and especially far-red light, are of particular interest (13). The longer wavelength light extends the range of fluorescence resonance energy transfer (FRET)-based applications and causes fewer damaging events to proteins and DNA because of its lower energy. The most favorable "optical window" for the visualization in living tissues is ϳ650 -1100 nm (23). Light with wavelength longer than 1100 nm is absorbed by water. Detection of fluorescence from proteins with emission peaks much shorter than 650 nm encounters the problem of interfering cellular autofluorescence. At present the brightest red fluorescent proteins have emission maxima too far from the preferred "optical window." Besides, their excitation maxima...

Monomeric red fluorescent proteins with a large Stokes shift

“…Using simultaneous site-specific mutagenesis we constructed a library, in which residues 69, 148, 165, and 167 were randomized. These positions were identified from the X-ray structure of mKate (13) and substitutions which were shown to affect photostability of TagRFP variants (14). Using a custom array of 36 high-power 455 nm light-emitting diodes, we photobleached 10 cm Petri dishes with bacterial colonies expressing the FP mutants and selected those which maintained the most fluorescence.…”

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states

“…The detected slight non-coplanarity may results from a mixture of the PAmCherry1 molecules in the crystal photoactivated to different extents. However, the photoactivated PAmCherry1 indeed has a slightly noncoplanar chromophore as it has been shown for several other RFPs such as Rtms5 (16) and KFP (19).…”

“…This hydrogen bond, together with the stacking interaction between the hydroxyphenyl group and positively charged Arg-197, are possibly the important determinants stabilizing the trans configuration of chromophore and its anionic fluorescent state. Phenolic ring of the trans chromophores in mKate (19) and Rtms5 (21) makes stacking interaction with the guanidinium group of Arg-197. Similar stacking with positively charged His-197 is important for efficient fluorescence of amFP486 (22), KFP (23), and eqFP611 (16).…”

Macromolecular complexes in crystals and solutions

“…1 shows a reasonably well packed homotetrameric complex which is easily identifiable in the crystal packing (PDB entry 3bxc). However, the structure has been found to be monomeric in solution (Pletnev et al, 2008).…”

“…Homotetrameric complex of far-red fluorescent mKate protein easily identifiable by visual inspection of crystal packing (PDB entry 3bxc). However, the protein is found to be monomeric in solution (Pletnev et al, 2008). The picture was produced using the CCP4mg graphical viewer (Potterton et al, 2004).…”