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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,(More)
We briefly review the current literature where optogenetics has been used to study various aspects of astrocyte physiology in vitro and in vivo. This includes both genetically engineered Ca(2+) sensors and effector proteins, such as channelrhodopsin. We demonstrate how the ability to target astrocytes with cell-specific viral vectors to express optogenetic(More)
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(More)
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(More)
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(More)
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,(More)
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(More)
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,(More)
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(More)
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(More)