A Semisynthetic Fluorescent Sensor Protein for Glutamate

Abstract

ABSTRACT:We report the semisynthesis of a fluorescent glutamate sensor protein on cell surfaces. Sensor excitation at 547 nm yields a glutamate-dependent emission spectrum between 550 and 700 nm that can be exploited for ratiometric sensing. On cells, the sensor displays a ratiometric change of 1.56. The high sensitivity toward glutamate concentration changes of the sensor and its exclusive extracellular localization make it an attractive tool for glutamate sensing in neurobiology.T he amino acid glutamate is the prevalent neurotransmitter in the vertebrate nervous system. It is used at well over 90% of the synapses in the human brain and influences essentially all forms of behavior, including consciousness, sensory perception, motor control, and mood. 1 Further, glutamate is involved at most synapses that are modifiable, that is, that are capable of adapting to changing patterns of stimuli by enhancing or reducing the efficiency of synaptic transmission. 2 These processes are thought to be responsible for high-order brain functions, such as learning and memory. Three fluorescent sensor proteins for investigating the role of glutamate in neurobiology have been developed so far. 3−5 However, the modest performance of these sensor proteins has limited their use. Here we present a semisynthetic fluorescent sensor protein for glutamate which shows higher sensitivity toward glutamate concentration changes and operates at longer wavelengths than the previously reported sensors.Semisynthetic fluorescent sensor proteins (Snifits), 6,7 are fusion proteins consisting of SNAP-tag, 8 CLIP-tag, 9 and an analyte-binding protein ( Figure 1A). SNAP-and CLIP-tag are labeled with a synthetic fluorescent ligand and a second synthetic fluorophore, respectively. The ligand binds to the binding protein in an intramolecular fashion and thereby keeps the sensor protein in a closed conformation. Free analyte can compete for binding to the binding protein and can shift the equilibrium to the open conformation. This shift can be detected by a change in the Forster resonance energy transfer (FRET) efficiency between the two fluorophores.For the construction of a glutamate sensor protein based on the Snifit sensor concept, we have chosen the ionotropic glutamate receptor 5 (iGluR5) as the binding protein for two reasons. First, due to the modular construction of ionotropic glutamate receptors, it is possible to express its glutamate binding domain S1S2 as a soluble protein in bacteria while conserving both the high affinity and specificity toward glutamate. 10,11 The possibility to characterize the soluble binding protein in vitro before its use on cell surfaces facilitates sensor development. Second, it is known that the stereoselective functionalization of the γ-carbon of the glutamate side chain does not significantly perturb its affinity toward iGluR5, 12,13 suggesting an attachment point for the required synthetic tether. We therefore prepared the tethered glutamate analogue 1 (Figure 1B, Scheme S1−S3) that contains a Cy5 fluorophore a...

Enhancement of TFO Triplex Formation by Conjugation with Pyrene <i>via</i> Click Chemistry

“…The amidite unit 13 was applied to an automated DNA/RNA solid phase synthesizer and incorporated into intra-oligodeoxynucleotides (intra-ODNs) targeting the survivin oncogene. 19,23) Then, the synthesized ODNs were reacted with corresponding azido-modified compounds (1-10) [24][25][26][27][28][29] under the Huisgen cycloaddition reaction conditions 30) on controlled pore glass (CPG). The CPG resins were washed with water and acetonitrile in succession.…”

Synthetic protein switches: design principles and applications

“…To this end, a new class of semisynthetic protein switches, termed SNAP-tag based indicator with a fluorescent intramolecular tether (SNIFITs), promises a generic route to fluorescent small molecule sensors [24][25][26][27]. SNIFITs feature a protein-based binder tethered to an intramolecular ligand that can be displaced by a target analyte, which causes the SNIFIT to transition from a compact to an extended conformation.…”

Engineering BRET-Sensor Proteins

“…In the Snifit sensors, the ligand-binding domain is genetically fused to a fluorescent donor protein or a CLIP-tag domain, followed by a linker and SNAP-tag domain ( Fig. 3A) (Brun et al, 2012;Brun, Tan, Nakata, Hinner, & Johnsson, 2009). The SNAP-and CLIP-tags are self-labeling protein domains that can be specifically functionalized with a molecule of interest.…”