A genetically encoded, high‐signal‐to‐noise maltose sensor

Marvin, Jonathan S.; Schreiter, Eric R.; Echevarría, Ileabett; Looger, Loren L.

doi:10.1002/prot.23118

Cited by 106 publications

(157 citation statements)

References 59 publications

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“…caused by a change in pH at the synapse, AAV.hSynapsin.iMaltSnFR 21 , which has a similar pH profile to that of iGluSnFR (Supplementary Figs. 9 & 18) was tested as well, and showed no response (Fig.…”

Section: Resultsmentioning

confidence: 99%

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An optimized fluorescent probe for visualizing glutamate neurotransmission

et al. 2013

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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 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 post-synaptic 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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Section: Resultsmentioning

confidence: 99%

“…We pioneered this technique using the E. coli maltose-binding protein MalE (MBP) 21 , and extended it to the E. coli phosphonate-binding protein PhnD 22 . In both cases, high-resolution X-ray crystal structures were available in both the ligand-free, open and the ligand-bound, closed conformations.…”

Section: Introductionmentioning

confidence: 99%

An optimized fluorescent probe for visualizing glutamate neurotransmission

et al. 2013

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“…iGluSnFR comprises a circularly permuted GFP linked to a bacterial glutamate/aspartate-binding protein, and has been used for long-term imaging of glutamate release in soma, dendrites and dendritic spines in worms, zebrafish, and mammals. 24,25 Compared with the FRET-based probes, iGluSnFR has high SNR and brightness, photostability, and fast kinetics (rise t 1/2 = 15 ms, decay t 1/2 = 92 ms). In situations where the glutamatergic activity arriving at a postsynaptic neuron is the parameter of interest, glutamate-sensor proteins hold promise as a tool for neuroscience.…”

Section: Neural Activity-dependent Probes: Past Present and Near mentioning

confidence: 99%

Fast calcium sensor proteins for monitoring neural activity

et al. 2014

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A major goal of the BRAIN Initiative is the development of technologies to monitor neuronal network activity during active information processing. Toward this goal, genetically encoded calcium indicator proteins have become widely used for reporting activity in preparations ranging from invertebrates to awake mammals. However, slow response times, the narrow sensitivity range of Ca2+ and in some cases, poor signal-to-noise ratio still limit their usefulness. Here, we review recent improvements in the field of neural activity-sensitive probe design with a focus on the GCaMP family of calcium indicator proteins. In this context, we present our newly developed Fast-GCaMPs, which have up to 4-fold accelerated off-responses compared with the next-fastest GCaMP, GCaMP6f. Fast-GCaMPs were designed by destabilizing the association of the hydrophobic pocket of calcium-bound calmodulin with the RS20 binding domain, an intramolecular interaction that protects the green fluorescent protein chromophore. Fast-GCaMP6f-RS06 and Fast-GCaMP6f-RS09 have rapid off-responses in stopped-flow fluorimetry, in neocortical brain slices, and in the intact cerebellum in vivo. Fast-GCaMP6f variants should be useful for tracking action potentials closely spaced in time, and for following neural activity in fast-changing compartments, such as axons and dendrites. Finally, we discuss strategies that may allow tracking of a wider range of neuronal firing rates and improve spike detection.

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“…Fluorescent indicators can be made from either coupled small molecule dyes 22 or by genetic fusion to fluorescent protein(s) 23 . We have recently published a method for creating high signal-to-noise single-wavelength sensors for small molecules from periplasmic binding proteins, using E. coli maltose-binding protein as a test case 24 . The technique involves insertion of circularly permuted variants of fluorescent proteins, e.g.…”

Section: Introductionmentioning

confidence: 99%

Structure of the Escherichia coli Phosphonate Binding Protein PhnD and Rationally Optimized Phosphonate Biosensors

Alicea

Marvin

Miklos

et al. 2011

Journal of Molecular Biology

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The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ~70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into phosphonate uptake by bacteria and facilitated the rational design of high signal-to-noise phosphonate biosensors based both on coupled small molecule dyes and autocatalytic fluorescent proteins.

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A genetically encoded, high‐signal‐to‐noise maltose sensor

Cited by 106 publications

References 59 publications

An optimized fluorescent probe for visualizing glutamate neurotransmission

An optimized fluorescent probe for visualizing glutamate neurotransmission

Fast calcium sensor proteins for monitoring neural activity

Structure of the Escherichia coli Phosphonate Binding Protein PhnD and Rationally Optimized Phosphonate Biosensors

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