Control of Protonated Schiff Base Excited State Decay within Visual Protein Mimics: A Unified Model for Retinal Chromophores

Artificial biomimetic chromophore-protein complexes inspired by natural visual pigments can feature color tunability across the full visible spectrum. However, control of excited state dynamics of the retinal chromophore, which is of paramount importance for technological applications, is lacking due to its complex and subtle photophysics/photochemistry. Here, ultrafast transient absorption spectroscopy and quantum mechanics/molecular mechanics simulations are combined for the study of highly tunable rhodopsin mimics, as compared to retinal chromophores in solution. Conical intersections and transient fluorescent intermediates are identified with atomistic resolution, providing unambiguous assignment of their ultrafast excited state absorption features. The results point out that the electrostatic environment of the chromophore, modified by protein point mutations, affects its excited state properties allowing control of its photophysics with same power of chemical modifications of the chromophore. The complex nature of such fine control is a fundamental knowledge for the design of bio-mimetic opto-electronic and photonic devices. A joint experimental-computational study elucidates the photophysics of retinal Schiff bases embedded in artificial proteins capable of mimicking the color tunability of natural visual pigments as compared to solvated chromophores. Combining ultrafast transient absorption spectroscopy with quantum mechanics/molecular mechanics simulations allowed monitoring nonlinear optical signals of the chromophore, shedding light on the complexity beyond the fine control of its excited state lifetime

Fluoride export (FEX) proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore

<div><p>The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F^-), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a ‘double-barreled’ channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant <i>Arabidopsis thaliana</i> and an animal <i>Amphimedon queenslandica</i>, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast <i>Saccharomyces cerevisiae</i>. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are ‘single-barreled’ transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.</p></div

Model of Fex1p from <i>S</i>. <i>cerevisiae</i>.

<p>Comparison of the residues in two pores for Fex1p-<i>Sc</i> (red residues are in N-terminal domain and blue are in C-terminal domain) and Fluc-<i>Bp</i> (white). Residues in Pore II and not Pore I of FEX are similar to bacterial Fluc.</p

Effect of mutations to residues in the hypothetical pores of Fex1p.

<p>Effect of mutations to residues in the hypothetical pores of Fex1p.</p

Growth rescue of <i>fex1Δfex2Δ</i> yeast expressing previously uncharacterized FEX-like genes.

<p>(A) The strains were grown on YPD and YPD containing 5 mM NaF to determine rescue phenotype. Plant <i>FEX-At</i> is a putative FEX gene from <i>A</i>. <i>thaliana</i> cloned into the p426GPD vector. Animal <i>FEX-Aq</i> is a putative FEX gene from <i>A</i>. <i>queenslandica</i> cloned into the p426GPD vector. Yeast rescue plasmid (pRS416-<i>FEX1</i>) was used as a positive control and empty vector (p426GPD) was used as a negative control. (B) Quantification of the growth tolerance to NaF of yeast strains described in A.</p

Structural arrangement of fluoride export proteins.

<p>(A) Topology model of bacterial Fluc protein. (B) Tertiary structure of Fluc-<i>Bp</i> (PDB ID: 5FXB) showing two pores with fluoride (dark grey) ions. Sodium ion (purple) is in the middle of the protein dimer. Two monomers are colored brown and light grey. (C) Arrangement of transmembrane helices (top view). Helices TM3b from different monomers (yellow and green) separate two pores and have residues belonging to two different pores but located on the same helix. (D) Topology model of eukaryotic FEX protein with proposed interactions between TM3 and TM8 based on Fluc crystal structure.</p

Effect of mutations at conserved residues in two domains of Fex1p.

<p>Effect of mutations at conserved residues in two domains of Fex1p.</p

Alignment of protein sequences from six eukaryotic and two bacterial fluoride channels.

<p>Transmembrane regions were predicted using TMHMM and TMpred servers. Conserved residues for all eukaryotic FEX proteins are highlighted with asterisks. Amino acid numbering corresponds to the protein sequence of Fex1p from <i>S</i>. <i>cerevisiae</i>. The residues highlighted in green are present within both pores of Fluc.</p

Functional analysis of conserved S/T fragments in domain 1 (red) and domain 2 (blue).

<p>Conserved Ser/Thr residues are located near putative Na⁺ binding site.</p

The helices from both domains that form functional and vestigial pores.

<p>The helices from both domains that form functional and vestigial pores.</p