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). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C(ß) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.

Near-infrared fluorescent proteins.

Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

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. An examination of the structures demonstrated that there are two groups of changes responsible for the bathochromic shift of excitation/emission bands of these proteins relative to their predecessor. The first group of changes resulted in an increase of hydrophilicity at the acylimine site of the chromophore due to the presence of one and three water molecules in eqFP650 and eqFP670, respectively. These water molecules provide connection of the chromophore with the protein scaffold via hydrogen bonds causing an ~15 nm bathochromic shift of the eqFP650 and eqFP670 emission bands. The second group of changes observed in eqFP670 arises from substitution of both Ser143 and Ser158 by asparagines. Asn143 and Asn158 of eqFP670 are hydrogen bonded with each other, as well as with the protein scaffold and with the p-hydroxyphenyl group of the chromophore, resulting in an additional ~20 nm bathochromic shift of the eqFP670 emission band as compared to eqFP650. The role of the observed structural changes was verified by mutagenesis.

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na+/Ca2+ exchange.

Fluctuating extracellular Ca2+ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca2+ from 1.8 to < or = 0.6 mM by chelation with EGTA induces a marked spike in O2 consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca2+, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na+ and the subsequent activation of mitochondrial Na+/Ca2+ and Na+/H+ exchange. The mitochondrial Na+/Ca2+ exchanger ((m)NCX) activated by Na+ influx reduces Ca2+ and increases Na+ levels in the mitochondrial matrix. The excess of Na+ activates the mitochondrial Na+/H+ exchanger (NHE) increasing the outward pumping of protons, electron transport and O2 consumption. Reduction in extracellular Na+ and inhibition of Na+ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the (m)NCX, L-type voltage gated Ca2+ channels or the release of Ca2+ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca2+ exchange is critical for response development.

The structure of Ca2+ sensor Case16 reveals the mechanism of reaction to low Ca2+ concentrations.

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca(2+) sensor, Case16, in the presence of a low Ca(2+) concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca(2+)-dependent response when only two out of four Ca(2+)-binding pockets of calmodulin (CaM) are occupied with Ca(2+) ions. In such a "half Ca(2+)-bound state", Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca(2+) sensor Case12 at saturating Ca(2+) concentration. Based on this structure, we postulate that cpGFP-based Ca(2+) sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca(2+) sensors in living cells.

Practical and reliable FRET/FLIM pair of fluorescent proteins.

BACKGROUND: In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings. RESULTS: Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter). CONCLUSION: The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.

Conversion of red fluorescent protein into a bright blue probe.

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.

Single fluorescent protein-based Ca2+ sensors with increased dynamic range.

BACKGROUND: Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). RESULTS: Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. CONCLUSION: We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

Photoswitchable cyan fluorescent protein for protein tracking.

In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing.

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio-temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore-assisted light inactivation (CALI) of proteins, as well as for photo-induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini-antibodies or selective scaffold proteins and photo-induced cytotoxicity of miniSOG, which is particularly promising for selective non-invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

Photoswitchable cyan fluorescent protein as a FRET donor.

Among a variety of fluorescent proteins available today, there is a lack of suitable markers with excitation/emission in the violet/blue part of visible spectrum. Recently, we reported on photoswitchable cyan fluorescent protein (PS-CFP), which represents monomeric high-contrasting photactivatable label for in vivo protein movement tracking. However, PS-CFP demands high intensity of light for the photoswitching. Therefore it can be employed as a common fluorescent tag at conventional light intensities, which cause negligible or zero photoactivation. High pH stability and unique positioning of excitation/emission peaks make it a worthy supplement to the existing palette of fluorescent proteins. Here we use PS-CFP fusion with a yellow fluorescent protein phiYFP to show that PS-CFP is a promising donor partner for the fluorescence resonance energy transfer (FRET). A remarkable phenomenon is that PS-CFP donor fluorescence turned to be essentially stable with and without FRET, while acceptor emission demonstrated record dynamic range of up to 7.8-fold. This makes the FRET pair presented a useful tool for the single color high throughput screenings. Here we also propose ways for further PS-CFP enhancing, aiming to develop bright cyan fluorescent protein with unique spectral characteristics.

Astroglia are a possible cellular substrate of angiotensin(1-7) effects in the rostral ventrolateral medulla.

AIMS: Angiotensin(1-7) (Ang1-7) acting at the level of the rostral ventrolateral medulla (RVLM) affects arterial pressure. The cellular substrate of Ang1-7 remains unknown. We sought to determine which cell types in RVLM could mediate its actions and whether these are altered in the spontaneously hypertensive rat (SHR). METHODS AND RESULTS: Astrocytes, catecholaminergic (CA-ergic) and non-CA-ergic neurones were targeted with adenoviral vectors in organotypic slice cultures from Wistar rats and SHR. Astrocytic Ca(2+) signalling was monitored using a genetically engineered Ca(2+) sensor Case12. CA-ergic neurones expressed enhanced green fluorescent protein (EGFP) under control of the PRS x 8 promoter, whereas non-CA-neurones expressed EGFP under control of the synapsin-1 promoter. Neurones were recorded in whole cell mode while [Ca(2+)](i) was monitored using Rhod-2. RVLM astrocytes responded to Ang1-7 (200-1000 nM) with concentration-dependent [Ca(2+)](i) elevation. In SHR, the response to 1000 nM was significantly attenuated. The competitive Ang1-7 receptor antagonist A779, but not the AT(1) receptor blocker (losartan), suppressed Ang1-7-induced [Ca(2+)](i) elevations, which were also antagonized by blocking intracellular Ca(2+) stores. Ang1-7 evoked no consistent changes in [Ca(2+)](i) or membrane excitability in CA-ergic or non-CA-ergic neurones in either rat strain. CONCLUSION: Astroglia are a plausible cellular target of Ang1-7 in RVLM. Our data suggest that astrocytic responsiveness to Ang1-7 is reduced in SHR. We hypothesise that Ang1-7 modulates astrocytic signalling which in vivo may affect local metabolism and microcirculation, resulting in changes in activity of RVLM pre-sympathetic neurones and hence blood pressure.

Recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis.

The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant "epitope library" covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48-70 and 85-170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43-68 and 146-170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81-103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.