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We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP‐like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high‐resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and E (c) 14,300 M(−1) cm(−1)) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

Graphical abstract, For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G – a variant of the enhanced yellow fluorescent protein – obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (~85%) and mature chromophore (~15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with ~ 2-fold improved brightness were engineered.

Super-resolution fluorescent imaging in living cells remains technically challenging due in large part to the photodecomposition of fluorescent tags. Recently suggested protein-PAINT is the only su...

The crystal structure of monomeric red fluorescent protein FusionRed (λex/λem 580/608 mn) has been determined at 1.09 A resolution and revealed two alternative routes of post-translational chemistry, resulting in distinctly different products. The refinement occupancies suggest the 60:40 ratio of the mature Met63-Tyr64-Gly65 chromophore and uncyclized chromophore-forming tripeptide with the protein backbone cleaved between Met63 and the preceding Phe62 and oxidized Cα-Cβ bond of Tyr64. We analyzed the structures of FusionRed and several related red fluorescent proteins, identified structural elements causing hydrolysis of the peptide bond, and verified their impact by single point mutagenesis. These findings advance the understanding of the post-translational chemistry of GFP-like fluorescent proteins beyond the canonical cyclization-dehydration-oxidation mechanism. They also show that impaired cyclization does not prevent chromophore-forming tripeptide from further transformations enabled by the same set of catalytic residues. Our mutagenesis efforts resulted in inhibition of the peptide backbone cleavage, and a FusionRed variant with ~30% improved effective brightness.

GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine, and efforts to replace it were in vain. Now, we have the first structure of a fluorescent protein with a successfully matured chromophore that has alanine as the third chromophore-forming residue. Consideration of the protein structures revealed two alternative routes of posttranslational transformation, resulting in either chromophore maturation or hydrolysis of GYG/GYA tripeptide. Both transformations are catalyzed by the same set of catalytic residues, Arg88 and Glu35-Wat-Glu211 cluster, whereas the residues in positions 62 and 102 shift the equilibrium between chromophore maturation and hydrolysis.

Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes. Once photobleached, the dye in the complex is replaced with a fresh fluorogen available in the sample. With suitable kinetics, this replacement creates fluorescence blinking required for attaining super-resolution and overcomes photobleaching associated with the loss of an irreplaceable fluorophore. Here we report on the rational design of two protein-PAINT tags based on the 1.58 Å crystal structure of the DiB1:M739 complex, an improved green-emitting DiB3/F74V:M739 and a new orange-emitting DiB3/F53L:M739. They outperform previously reported DiB-based tags to become best in class biomarkers for protein-PAINT. The new tags advance protein-PAINT from the proof-of-concept to a reliable tool suitable for prolonged super-resolution imaging of intracellular proteins in fixed and living cells and two-color PAINT-like nanoscopy with a single fluorogen.

The influence of lipophilic, electrically neutral surfactants added to the membrane on the ion transfer resistance between an aqueous sample and a polymeric ion-selective membrane has been studied by electric impedance spectroscopy and potentiometry. An increase in the surface resistance and a shift of the apparently super-Nernstian response to lower sample ion activities has been observed when using the nonpolar bis(2-ethylhexyl) sebacate as plasticizer.

Multimode PMMA optical fibres with a cladding made from ion-selective membrane were designed, prepared, and investigated. Some were prepared by direct drawing of membrane-coated preforms and others were overcoated. Results obtained indicate loss of membrane sensitivity that is attributed to decrease of its ionic permeability. Absorbance of the fibre follows a modified Beer law as confirmed by rank analysis of series of spectra and explained by significant mode coupling within the fibre.

In a novel biosensing approach, a stearyl-[small beta]-d-glucopyranoside layer is formed by self-organization at the interface between a solvent polymeric membrane and the aqueous sample phase and its interaction with concanavalin A is detected by electric impedance spectroscopy.