Crystal Structure of Bright Fluorescent Protein BrUSLEE with Subnanosecond Fluorescence Lifetime; Electric and Dynamic Properties.

The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component of fluorescence lifetime (FL~0.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

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.

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design.

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.

Structure-Based Rational Design of Two Enhanced Bacterial Lipocalin Tags for Protein-PAINT Super-resolution Microscopy.

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.

Two independent routes of post-translational chemistry in fluorescent protein FusionRed.

The crystal structure of monomeric red fluorescent protein FusionRed (λ/λ 580/608 mn) has been determined at 1.09 Å 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.

Structural Factors Enabling Successful GFP-Like Proteins with Alanine as the Third Chromophore-Forming Residue.

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.

Structural insights on starch hydrolysis by plant β-amylase and its evolutionary relationship with bacterial enzymes.

The conversion of starch to maltose is catalysed in plants by β-amylase. The enzymatic mechanism has been well-characterized for the soybean and barley enzymes, which utilise a glutamic acid-glutamate pair. In the present study, we present a surprise observation of maltotetraose at the active site, the presence of which elucidates the clear role of Thr344 as a conformational "switch" between substrate binding and product release during hydrolysis.

Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms.

The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm).

Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore.

Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.

Structure of the green fluorescent protein NowGFP with an anionic tryptophan-based chromophore.

A green-emitting fluorescent variant, NowGFP, with a tryptophan-based chromophore (Thr65-Trp66-Gly67) was recently developed from the cyan mCerulean by introducing 18 point mutations. NowGFP is characterized by bright green fluorescence at physiological and higher pH and by weak cyan fluorescence at low pH. Illumination with blue light induces irreversible photoconversion of NowGFP from a green-emitting to a cyan-emitting form.

[Three dimensional structure of the dimeric gene-engineered variant of green fluorescent protein EGFP-K162Q in P6(1) crystal space group].

The crystal structure of the dimeric green fluorescent protein EGFP-K162Q with C-terminal deletion MDELYK (EGFPv) has been determined in space group P6 at resolution 1.34 A. The obtained structure has been compared with that of the monomeric form of EGFP (green biomarker with enhanced photophysical properties) determined in other crystal space group P2(1)2(1)2(1) at resolution 1.

Observations and lessons learnt from non health professionals evaluating a health search engine.

This article presents the results of one of the stages of the user-centered evaluation conducted in a framework of the EU project Khresmoi. In a controlled environment, users were asked to perform health-related tasks using a search engine specifically developed for trustworthy online health information. Twenty seven participants from largely the Czech Republic and France took part in the evaluation.

General vs health specialized search engine: a blind comparative evaluation of top search results.

This paper presents the results of a blind comparison of top ten search results retrieved by Google.ch (French) and Khresmoi for everyone, a health specialized search engine. Participants--students of the Faculty of Medicine of the University of Geneva had to complete three tasks and select their preferred results.

Orange fluorescent proteins: structural studies of LSSmOrange, PSmOrange and PSmOrange2.

A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (λex/λem at 437/572 nm), PSmOrange (λex/λem at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (λex/λem at 546/561 nm and for photoconverted form at 619/651 nm), is presented. The obtained crystallographic structures provide an understanding of how the ensemble of a few key mutations enabled special properties of the orange FPs. While only a single Ile161Asp mutation, enabling excited state proton transfer, is critical for LSSmOrange, other substitutions provide refinement of its special properties and an exceptional 120 nm large Stokes shift.

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria).

Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis.

The yellow fluorescent protein phiYFPv (λem(max) ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow-orange range (535-555 nm). Here, the crystal structure of phiYFPv has been determined at 2.

Structural basis for bathochromic shift of fluorescence in far-red fluorescent proteins eqFP650 and eqFP670.

The crystal structures of the far-red fluorescent proteins (FPs) eqFP650 (λ(ex)(max)/λ(em)(max) 592/650 nm) and eqFP670 (λ(ex)(max)/λ(em)(max) 605/670 nm), the successors of the far-red FP Katushka (λ(ex)(max)/λ(em)(max) 588/635 nm), have been determined at 1.8 and 1.6 Å resolution, respectively.

Online health information search: what struggles and empowers the users? Results of an online survey.

The most popular mean of searching for online health content is a general search engine for all domains of interest. Being general implies on one hand that the search engine is not tailored to the needs which are particular to the medical and on another hand that health domain and health-specific queries may not always return adequate and adapted results. The aim of our study was to identify difficulties and preferences in online health information search encountered by members of the general public.

[Catalytic sites of medicinal leech enzyme destabilase-lysozyme (Mldl). Structure-functional correlation].

Based on three-dimensional model of the bifunctional enzyme Destabilase-Lysozyme (mlDL-Ds2) in complex with trimer of N-acetylglucosoamine (NAG)3 the functional role of the stereochemically based group of amino acids (Glu14, Asp26, Ser 29, Ser31, Lys38, His92), in manifestation of glycosidase and isopeptidase activities has been elucidated. By method of site-directed mutagenesis it has been shown that mlDL glycosidase active site includes catalytic Glu14 and Asp26, and isopeptidase site functions as Ser/Lys dyad presented by catalytic residues Lys38 and Ser29. Thus, among the invertebrate lysozymes mlDL presents first example of the bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.

Results of the 10 HON survey on health and medical internet use.

The Internet is increasingly being used as a means to search and communicate health information. As the mission of Health on the Net Foundation (HON) is to guide healthcare consumers and professionals to trustworthy online information, we have been interested in seeing the trend of the attitudes towards Internet use for health purposes since 1996. This article presents the results of the 10 HON survey conducted in July-August 2010 (in English and French).

Crystallographic study of red fluorescent protein eqFP578 and its far-red variant Katushka reveals opposite pH-induced isomerization of chromophore.

The wild type red fluorescent protein eqFP578 (from sea anemone Entacmaea quadricolor, λ(ex) = 552 nm, λ(em) = 578 nm) and its bright far-red fluorescent variant Katushka (λ(ex) = 588 nm, λ(em) = 635 nm) are characterized by the pronounced pH dependence of their fluorescence. The crystal structures of eqFP578f (eqFP578 with two point mutations improving the protein folding) and Katushka have been determined at the resolution ranging from 1.15 to 1.

Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis.

The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence (lambda(ex) = 480 nm, lambda(em) = 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure.

Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed.

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants.

A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore.

The far-red fluorescent protein mKate (lambda(ex), 588 nm; lambda(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.

Pixelated liquid-crystal light valve for neural network application.

The liquid-crystal light valve (LCLV) is a useful component for performing integration, thresholding, and gain functions in optical neural networks. Integration of the neural activation channels is implemented by pixelation of the LCLV, with use of a structured metallic layer between the photoconductor and the liquid-crystal layer. Measurements are presented for this type of valve, examples of which were prepared for two specific neural network implementations.