Optimized and Far-Red-Emitting Variants of Fluorescent Protein eqFP611

Abstract

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

“…12 Indeed, the residue corresponding to Glu16 in mPlum is hydrophobic in all known red fluorescent proteins, that is valine, leucine, isoleucine, and phenolalanine. This is also the case for the more recently derived far-red fluorescent proteins mKate (k em ¼ 635 nm) 24 and RFP639 (k em ¼ 639 nm). 24 As the proton acceptor is conjugated with the rest of the chromophore, it is reasonable to assume that the interaction is capable of significantly affecting the electronic configuration of the chromophore.…”

“…This is also the case for the more recently derived far-red fluorescent proteins mKate (k em ¼ 635 nm) 24 and RFP639 (k em ¼ 639 nm). 24 As the proton acceptor is conjugated with the rest of the chromophore, it is reasonable to assume that the interaction is capable of significantly affecting the electronic configuration of the chromophore. Furthermore, it has long been established that hydrogen bonds are directional interactions so that the details of distance and orientation could have important consequences for excitation and emission spectra.…”

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries

“…For example, these proteins have been used as markers of gene expression (1), expressed as fusions to track endogenous protein within cells (2), and applied with other fluorescent proteins (FPs) for use in FRET experiments (3). The availability of monomeric versions of these proteins has bolstered their worth as fusion tags and vaulted them into routine experimental use (4)(5).…”

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

“…Molecular phylogeny showed that the colors evolved from a single, most likely green fluorescent ancestral protein by variation of the p-HBI chromophore (19,23). The color palette was extended considerably, especially toward the far-red end of the spectrum by protein engineering (Table 1) (24)(25)(26)(27). The chromophore structures and fluorescence spectra of the major color classes are shown in Fig.…”

“…In mPlum, the red shift is presumably induced by bringing the carbonyl oxygen of the amino acid preceding the first chromogenic residue into a coplanar arrangement with the chromophore (55). The red shift of RFP639 is likely caused by optimized p-stacking interactions of His197 and the phenyl group of the chromogenic tyrosine as a result of a trans-cis isomerisation of the chromophore (24,56).…”