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Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.
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Sinalização do Cálcio , Cálcio , Calmodulina , Neurônios , Óxido Nítrico Sintase Tipo III , Fragmentos de Peptídeos , Cálcio/análise , Cálcio/metabolismo , Calmodulina/metabolismo , Neurônios/metabolismo , Cinética , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fatores de Tempo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.
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Ácido Glutâmico , Transmissão Sináptica , Camundongos , Animais , Ácido Glutâmico/metabolismo , Cinética , Neurônios/fisiologia , Sinapses/fisiologiaRESUMO
Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.
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Cálcio/análise , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Drosophila , Feminino , Lasers , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular , Córtex Somatossensorial/químicaRESUMO
Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.
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Corantes Fluorescentes/química , Hidrolases/química , Potenciais de Ação/efeitos dos fármacos , Animais , Bioengenharia , Cálcio/química , Células Cultivadas , Cristalografia por Raios X , Fenômenos Eletrofisiológicos , Corantes Fluorescentes/síntese química , Hidrolases/síntese química , Cinética , Conformação Molecular , Estrutura Molecular , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Proteínas/química , Ratos , RodaminasRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.
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Córtex Cerebral/fisiologia , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Tomografia/métodos , Animais , Cálcio/metabolismo , Córtex Cerebral/citologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Fótons , RatosRESUMO
We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators.
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Cálcio/química , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Biliverdina/química , DNA/análise , Escherichia coli/química , Feminino , Transferência Ressonante de Energia de Fluorescência , Vetores Genéticos , Células HeLa , Hipocampo/química , Humanos , Íons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Neurônios/química , Optogenética , Domínios ProteicosRESUMO
Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.
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Cálcio/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Drosophila , Feminino , Proteínas de Fluorescência Verde , Camundongos , Junção Neuromuscular/diagnóstico por imagem , Ratos , Córtex Visual/metabolismoRESUMO
In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.
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Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
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Potenciais de Ação , Proteínas de Ligação ao Cálcio/química , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Espinhas Dendríticas/metabolismo , Neurônios GABAérgicos/metabolismo , Proteínas Luminescentes/genética , Camundongos , Imagem Molecular , Mutagênese , Engenharia de Proteínas , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Córtex Visual/citologia , Córtex Visual/fisiologiaRESUMO
BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.
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Cálcio/análise , Substâncias Luminescentes/análise , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Animais , Células Cultivadas , Cristalografia/métodos , Células HeLa , Humanos , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Camundongos , Técnicas de Cultura de Órgãos , Estrutura Secundária de Proteína , Ratos , Anêmonas-do-Mar , Peixe-Zebra , Proteína Vermelha FluorescenteRESUMO
KEY POINTS: The genetically encoded fluorescent calcium integrator calcium-modulated photoactivatable ratiobetric integrator (CaMPARI) reports calcium influx induced by synaptic and neural activity. Its fluorescence is converted from green to red in the presence of violet light and calcium. The rate of conversion - the sensitivity to activity - is tunable and depends on the intensity of violet light. Synaptic activity and action potentials can independently initiate significant CaMPARI conversion. The level of conversion by subthreshold synaptic inputs is correlated to the strength of input, enabling optical readout of relative synaptic strength. When combined with optogenetic activation of defined presynaptic neurons, CaMPARI provides an all-optical method to map synaptic connectivity. ABSTRACT: The calcium-modulated photoactivatable ratiometric integrator (CaMPARI) is a genetically encoded calcium integrator that facilitates the study of neural circuits by permanently marking cells active during user-specified temporal windows. Permanent marking enables measurement of signals from large swathes of tissue and easy correlation of activity with other structural or functional labels. One potential application of CaMPARI is labelling neurons postsynaptic to specific populations targeted for optogenetic stimulation, giving rise to all-optical functional connectivity mapping. Here, we characterized the response of CaMPARI to several common types of neuronal calcium signals in mouse acute cortical brain slices. Our experiments show that CaMPARI is effectively converted by both action potentials and subthreshold synaptic inputs, and that conversion level is correlated to synaptic strength. Importantly, we found that conversion rate can be tuned: it is linearly related to light intensity. At low photoconversion light levels CaMPARI offers a wide dynamic range due to slower conversion rate; at high light levels conversion is more rapid and more sensitive to activity. Finally, we employed CaMPARI and optogenetics for functional circuit mapping in ex vivo acute brain slices, which preserve in vivo-like connectivity of axon terminals. With a single light source, we stimulated channelrhodopsin-2-expressing long-range posteromedial (POm) thalamic axon terminals in cortex and induced CaMPARI conversion in recipient cortical neurons. We found that POm stimulation triggers robust photoconversion of layer 5 cortical neurons and weaker conversion of layer 2/3 neurons. Thus, CaMPARI enables network-wide, tunable, all-optical functional circuit mapping that captures supra- and subthreshold depolarization.
Assuntos
Mapeamento Encefálico/métodos , Encéfalo/fisiologia , Potenciais de Ação , Animais , Feminino , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Optogenética , Estimulação Luminosa , Transmissão SinápticaRESUMO
We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.
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Proteínas de Escherichia coli , Corantes Fluorescentes , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde , Proteínas Recombinantes de Fusão , Transmissão Sináptica/fisiologia , Animais , Astrócitos/metabolismo , Técnicas Biossensoriais , Caenorhabditis elegans , Sinalização do Cálcio/fisiologia , Proteínas de Escherichia coli/síntese química , Potenciais Pós-Sinápticos Excitadores/fisiologia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/síntese química , Hipocampo/metabolismo , Camundongos , Córtex Motor/metabolismo , Neurônios/metabolismo , Estimulação Luminosa , Células Piramidais/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Retina/fisiologia , Razão Sinal-Ruído , Peixe-ZebraRESUMO
Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery.
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The phenotypical severity of sickle-cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α2ßs2) to contain embryonic ζ-globin in place of adult α-globin subunits (Hb ζ2ßs2). Crystallographical analyses of liganded Hb ζζ2ßs2, though, demonstrate a tense (T-state) quaternary structure that paradoxically predicts its participation in--rather than its exclusion from--pathological deoxyHb S polymers. We resolved this structure-function conundrum by examining the effects of αâζ exchange on the characteristics of specific amino acids that mediate sickle polymer assembly. Superposition analyses of the ßs subunits of T-state deoxyHb α2ßs2 and T-state CO-liganded Hb ζ2ßs2 reveal significant displacements of both mutant ßsVal6 and conserved ß-chain contact residues, predicting weakening of corresponding polymer-stabilizing interactions. Similar comparisons of the α- and ζ-globin subunits implicate four amino acids that are either repositioned or undergo non-conservative substitution, abrogating critical polymer contacts. CO-Hb ζ2ßs2 additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological ßsVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ2ßs2 suggests that it does not co-assemble with deoxyHb S in vivo. In sum, the antipolymer activities of Hb ζ2ßs2 appear to arise from both repositioning and replacement of specific α- and ßs-chain residues, favoring an alternate T-state solution structure that is excluded from pathological deoxyHb S polymers. These data account for the antipolymer activity of Hb ζ2ßs2, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.
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Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.
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Proteínas de Bactérias/química , Fases de Leitura Aberta , Palmitoil Coenzima A/química , Photobacterium/enzimologia , Multimerização Proteica/fisiologia , Tioléster Hidrolases/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Palmitoil Coenzima A/metabolismo , Estrutura Quaternária de Proteína , Especificidade por Substrato/fisiologiaRESUMO
Brain oscillations are crucial for perception, memory, and behavior. Parvalbumin-expressing (PV) interneurons are critical for these oscillations, but their population dynamics remain unclear. Using voltage imaging, we simultaneously recorded membrane potentials in up to 26 PV interneurons in vivo during hippocampal ripple oscillations in mice. We found that PV cells generate ripple-frequency rhythms by forming highly dynamic cell assemblies. These assemblies exhibit rapid and significant changes from cycle to cycle, varying greatly in both size and membership. Importantly, this variability is not just random spiking failures of individual neurons. Rather, the activities of other PV cells contain significant information about whether a PV cell spikes or not in a given cycle. This coordination persists without network oscillations, and it exists in subthreshold potentials even when the cells are not spiking. Dynamic assemblies of interneurons may provide a new mechanism to modulate postsynaptic dynamics and impact cognitive functions flexibly and rapidly.
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Interneurônios , Parvalbuminas , Animais , Parvalbuminas/metabolismo , Interneurônios/fisiologia , Camundongos , Hipocampo/fisiologia , Hipocampo/citologia , Potenciais de Ação/fisiologia , Encéfalo/fisiologia , Encéfalo/citologia , Camundongos Transgênicos , Ondas Encefálicas/fisiologia , MasculinoRESUMO
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
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Sinalização do Cálcio , Corantes Fluorescentes/química , Fluorometria/métodos , Proteínas de Fluorescência Verde/química , Neuroimagem/métodos , Neurônios/química , Peptídeos/química , Transmissão Sináptica , Animais , Astrócitos/química , Astrócitos/ultraestrutura , Caenorhabditis elegans , Cristalografia por Raios X , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Corantes Fluorescentes/análise , Genes Sintéticos , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Células HEK293/química , Células HEK293/ultraestrutura , Hipocampo/química , Hipocampo/citologia , Humanos , Larva , Lasers , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Junção Neuromuscular/química , Junção Neuromuscular/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Neurópilo/química , Neurópilo/fisiologia , Neurópilo/ultraestrutura , Neurônios Receptores Olfatórios/química , Neurônios Receptores Olfatórios/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Peptídeos/análise , Peptídeos/genética , Estimulação Luminosa , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Células Bipolares da Retina/química , Células Bipolares da Retina/fisiologia , Células Bipolares da Retina/ultraestrutura , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
A variant Hb ζ2ß2(s) that is formed from sickle hemoglobin (Hb S; α2ß2(s)) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2ß2(s) inhibits the polymerization of deoxygenated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2ß2), Hb ζ2ß2(s) exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95â Å resolution crystal structure of human Hb ζ2ß2(s) that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2ß2(s) displays a central water cavity, a ζ1-ß(s)2 (or ζ2-ß(s)1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal ßHis146 salt-bridge interactions, and a ß-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1ß(s)1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2ß2(s) and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.
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Anemia Falciforme/sangue , Anemia Falciforme/genética , Hemoglobina Falciforme/genética , Globinas zeta/química , Adulto , Regulação Alostérica/genética , Animais , Cristalografia por Raios X , Variação Genética , Hemoglobina Falciforme/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Oxigênio/metabolismo , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , alfa-Globinas/genética , alfa-Globinas/metabolismo , Globinas zeta/metabolismoRESUMO
Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.