Engineering a uniquely reactive thiol into a cysteine-rich peptide

Shimony, Ethan; Sun, Tsoyue; Kolmakova-Partensky, Ludmilla; Miller, Christopher

doi:10.1093/protein/7.4.503

Cited by 31 publications

(29 citation statements)

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“…As an alternate strategy for chemoselective conjugation, we revealed a free "spinster" thiol on the surface of the peptide (43). Removal of the Acm protecting group has typically been catalyzed by Ag + and facilitated with excess reductant (44), or oxidant (45), but these conditions are incompatible with preserving existing disulfides while obtaining a reduced surface thiol.…”

Section: Resultsmentioning

confidence: 99%

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Tilley

Eum

Fletcher-Taylor

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

111

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Significance Electrically excitable cells, such as neurons, exhibit tremendous variation in their patterns of electrical signals. These variations arise from the collection of ion channels present in any specific cell, but understanding which ion channels are at the root of particular electrical signals remains a significant challenge. Here, we describe novel probes, derived from a tarantula venom peptide, that are able to report the activity of voltage-gated ion channels in living cells. This technology uses state-selective binding to optically monitor the activation of ion channels during cellular electrical signaling. Activity-reporting probes based on these prototypes could potentially identify when endogenous ion channels contribute to electrical signaling, thus facilitating the identification of ion channel targets for therapeutic drug intervention.

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Section: Resultsmentioning

confidence: 99%

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Tilley

Eum

Fletcher-Taylor

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

111

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“…To tether the maleimide to CTX, we first synthesized the cleavable bismaleimide 1b by reacting bis(chlorocarbonyl)disulfane (13,14) with a 3-maleimido-N-[2-(phenylamino)ethyl]propanamide [see supporting information (SI) Materials and Methods]. CTXClv was then synthesized by adding excess 1b to the CTX mutant R19C (Materials and Methods), which upon derivatization with cysteine-modifying reagents has been shown to maintain its high affinity for the ion conduction pore of K ϩ channels (15). To specifically target and modify Q1/E1 complexes with CTX-Clv, we used a variant of Q1 that binds CTX with nanomolar affinity (6) and placed a single cysteine in the N terminus of E1 (T14C) to react with the maleimide.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant CTX R19C was purified and the methanethiosulfonate ethyltrimethylammonium (MTSET)-protected disulfide of CTX R19C was prepared as described in ref. 15. CTX-Clv was synthesized as follows: 16 nmol of CTX-MTSET in 2 ml of low-salt buffer (10 mM NaCl, 10 mM KPi, pH 7.4) was reduced with 1 mM dithiothreitol (DTT) for 45 min.…”

Section: Methodsmentioning

confidence: 99%

Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes

Morin

Kobertz

2008

Proc. Natl. Acad. Sci. U.S.A.

113

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Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting ␣-subunits with specific regulatory ␤-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of ␣-and ␤-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K ؉ channel inhibitor and its iterative application to tally the number of ␤-subunits in a KCNQ1/KCNE1 K ؉ channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K ؉ channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE ␤-subunit modulation of voltage-gated K ؉ channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K ؉ channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE ␤-subunits.membrane proteins that open and close in response to changes in membrane potential. To regulate cellular potassium flow in both excitable and nonexcitable tissues, Q1 channels coassemble with regulatory KCNE peptides, forming membraneembedded K ϩ channel complexes with various voltage-sensing and gating properties (1). The importance of forming a properly assembled Q1-KCNE complex is underscored by the mutations that give rise to long QT syndrome and congenital hearing loss (2, 3). Although the fourfold arrangement of the Q1 ␣-subunits along the ion conduction pathway is established and unquestioned, the number of KCNE ␤-subunits in the K ϩ channel complex has been a long-standing and heated debate (4-7). Goldstein and coworkers (6) have strongly argued that Q1 channels average two KCNE peptides per complex; however, they could not definitively rule out K ϩ channel complexes containing a solitary KCNE peptide. Another complicating factor is the notion that Q1 channels form complexes with KCNE ␤-subunits with multiple stoichiometries (5, 8). Thus, approaches that measure the average number of KCNE peptides in the Q1 channel complex at the cell surface are unable to readily discern between fixed and various stoichiometries.To determine the stoichiometry or stoichiometries of Q1-KCNE K ϩ channel complexes, we devised an iterative cell surface modification scheme where the labeling reagent specifically binds to the outer vestibule of the K ϩ conduction pore while covalently modifying a cysteine-bearing KCNE peptide in the channel complex (Fig. 1). Once th...

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“…The latter methodology permits direct incorporation of non-native amino acids during linear synthesis of the peptide as illustrated here by substitution of Lys-ε-d-LCbiotin at position 19 of the IbTx sequence. Biotin tags or fluorescent labels may also be coupled to peptide toxins by appropriate chemical modification reactions [(e.g., alkylation of the thiol group of a spinster cysteine (Shimony et al, 1994)] after synthesis and oxidation of paired disulfides. However, a practical coupling strategy requires high-yield labeling reactions and efficient purification of the bio-active derivative.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

et al. 2006

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Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca 2+ -activated K + channels with high affinity and specificity. Automated solid phase synthesis was used to prepare a biotin-labeled derivative (IbTx-LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N-ε-(d-biotin-6-amidocaproate)-L-lysine. Both IbTx-LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel. IbTx-LC-biotin exhibits high affinity (K D = 26 nM) and a slow dissociation rate (k off = 5.4 × 10 -4 s -1 ) in a macroscopic blocking assay of whole-cell current of the cloned human BK channel. Titration of IbTx-LC-biotin with StrAv monitored by high performance size exclusion chromatography is consistent with a stoichiometry of two binding sites for IbTx-LCbiotin per StrAv tetramer, indicating that steric interference hinders simultaneous binding of two toxin molecules on each of the two biotin-binding faces of StrAv. In combination with fluorescent conjugates of StrAv or anti-biotin antibody, IbTx-LC-biotin was used to image the surface distribution of BK channels on a transfected cell line. Fluorescence microscopy revealed a patchlike surface distribution of BK channel protein. The results support the feasibility of using IbTx-LCbiotin and similar biotin-tagged K + channel toxins for diverse applications in cellular neurobiology.

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Engineering a uniquely reactive thiol into a cysteine-rich peptide

Cited by 31 publications

References 0 publications

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

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Engineering a uniquely reactive thiol into a cysteine-rich peptide

Cited by 31 publications

References 0 publications

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Counting membrane-embedded KCNE β-subunits in functioning K + channel complexes

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca2+-Activated K+ Channel Expressed in a Human Cell Line

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Product

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Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line