Total Chemical Synthesis of Biologically Active Fluorescent Dye‐Labeled Ts1 Toxin

Dang, Bobo; Kubota, Tomoya; Correa, Ana M.; Bezanilla, Francisco; Kent, Stephen B. H.

doi:10.1002/ange.201404438

Cited by 7 publications

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References 23 publications

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“…Nor did the folding conditions we had previously developed for Ts1. [12] Addition of 20 % v/v DMSO to the redox buffer gave acceptable yields of discrete folded products of mass 8Dal ess than that of the corresponding polypeptide chains,w hich indicates the formation of four disulfide bonds.A fter careful optimization, the final folding conditions used were:[ polypeptide] 0.02 mg mL À1 ;2 .0 mm GSH/1.0 mm GSSG;0 .5 ml -arginine·HCl;1 .0 mm EDTA; 0.1m Tr is,2 0% DMSO,p H8.5;r oom temperature.D ata for the folding of Ts3(1-64)-CONH 2 are shown in Figure 2b. Using these conditions,w ew ere able to fold all four Ts3 candidate polypeptide chains,with concomitant formation of 4d isulfides in each case as shown by the loss of 8Da.…”

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Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

et al. 2016

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Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus. Ts3 binds to the domain IV voltage sensor of voltage-gated sodium channels (Nav) and slows dow n their fast inactivation. The covalent structure of Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Here we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure-activity studies that enabled us to establish the covalent structure of biologically active Ts3 protein molecule. We also report synthesis of the mirror image form of Ts3 toxin protein, and the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X-ray diffraction.

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Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

et al. 2016

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“…Although isolated from the same scorpion venom, Ts3 and Ts1 toxins are very different protein molecules:t hey have distinct biological activities-Ts1 is ab eta scorpion toxin while Ts3 is an alpha scorpion toxin; there is little sequence homology between the two polypeptide chains (only 16/53 conserved non-Cys residues; Figure 1) and the Ts3 polypeptide chain has much greater structural ambiguity at its Cterminus than was the case with the Ts1 toxin. [12] Although the Ts3 and Ts1 toxins do share aconserved pattern of eight Cys residues in the amino acid sequence of their polypeptide chains (Figure 1), it has been shown that, even where the pattern of Cys residues is conserved, scorpion venom proteins can have distinct tertiary structures. [13] Total chemical synthesis of the candidate Ts3 polypeptide chains was carried out by using amodular approach as shown in Scheme 1b.N ative chemical ligation (NCL) sites were chosen at -Tyr 23 -Cys 24 -a nd -Tyr 46 -Cys 47 -s ot hat, after preparation of the C-terminal peptide variants,t he peptide segments used were of similar length (23;2 3; and 16, 18, 20, or 21 amino acids).…”

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“…We have previously reported total synthesis of the Ts1 toxin protein molecule, [12] but that experience was of limited use for the current work. Although isolated from the same scorpion venom, Ts3 and Ts1 toxins are very different protein molecules:t hey have distinct biological activities-Ts1 is ab eta scorpion toxin while Ts3 is an alpha scorpion toxin; there is little sequence homology between the two polypeptide chains (only 16/53 conserved non-Cys residues; Figure 1) and the Ts3 polypeptide chain has much greater structural ambiguity at its Cterminus than was the case with the Ts1 toxin.…”

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confidence: 99%

“…[12] Addition of 20 % v/v DMSO to the redox buffer gave acceptable yields of discrete folded products of mass 8Dal ess than that of the corresponding polypeptide chains,w hich indicates the formation of four disulfide bonds.A fter careful optimization, the final folding conditions used were:[ polypeptide] 0.02 mg mL À1 ;2 .0 mm GSH/1.0 mm GSSG;0 .5 ml -arginine·HCl;1 .0 mm EDTA; 0.1m Tr is,2 0% DMSO,p H8.5;r oom temperature.D ata for the folding of Ts3(1-64)-CONH 2 are shown in Figure 2b. Nor did the folding conditions we had previously developed for Ts1.…”

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confidence: 99%

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Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

et al. 2016

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Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus.T s3 binds to the domain IV voltage sensor of voltage-gated sodium channels (Na v )and slows down their fast inactivation. The covalent structure of the Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Herein, we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure-activity studies that enabled us to establish the covalent structure of biologically active Ts3 toxin. We also report the synthesis of the mirror image form of the Ts3 protein molecule,a nd the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X-ray diffraction.

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“…Labelling of Cn2 [Q54Pra] was carried out via a slightly modified procedure, published previously (241…”

Section: Click-chemistry Labeling Of Cn2 [Q54pra] With Bodipymentioning

confidence: 99%

Elucidating the role of NaV1.6 in peripheral sensory neurons

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Chronic pain is estimated to affect 1 in 5 Australians. The condition is debilitating for patients and remains a costly burden on the healthcare system. Analgesics currently in clinical use lack efficacy, have low tolerability and are hampered with wide side-effect profiles. Therefore, the underlying molecular mechanisms of pain sensing must be elucidated with a view to the development of improved therapeutics. Peripheral sensory neurons are the site of pain signal initiation and are critical for the detection of painful stimuli. Neuronal excitability relies on integral membrane bound proteins such as voltage-gated sodium channels (NaV), which allow the influx of sodium and action potential propagation in these cells. Nine different NaV subtypes have been identified (NaV1.1-1.9) of which a subset are expressed in peripheral sensory neurons. The exact role that each NaV isoform plays in the excitability of different fibre types remains unclear. This thesis uses a multidisciplinary approach to assess the role of the NaV subtype NaV1.6 in peripheral sensory neurons.NaV1.6 channels share high sequence homology with other NaV isoforms expressed in peripheral sensory neurons. Scorpion venom is a rich source of bioactive compounds, many of which act on mammalian NaV channels. One such peptide, Cn2, is reported to be a selective NaV1.6 activator. Chapter 2 describes the chemical synthesis of Cn2 using a strategy of native chemical ligation. The pharmacology of the synthetically derived Cn2 is in line with that of the venom derived peptide. This approach allows the development of a number of probes, including a novel NaV1.6 antagonist Cn2[E15R], that retain selectivity for NaV1.6. Therefore, this chapter characterises novel NaV1.6 selective compounds and provides insight into NaV1.6-toxin interactions.Peripheral sensory neurons express a range of different ion channels that are critical for their function. Chapter 3 aimed to assess the contribution of NaV1.6 to Na + currents in mouse isolated dorsal root ganglion cells. High-content calcium imaging and whole-cell patch clamp electrophysiology was performed using the selective Cn2 peptide as a probe for NaV1.6 function. Corresponding to expression of NaV1.6, Cn2 induces early Na + currents in large but not small diameter DRG neurons. The Cn2 effect is absent in NaV1.6 -/neurons confirming the selectivity of the peptide in a neuronal environment. Furthermore, Cn2 enhances excitability of large diameter neurons in vitro.

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Total Chemical Synthesis of Biologically Active Fluorescent Dye‐Labeled Ts1 Toxin

Cited by 7 publications

References 23 publications

Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

Elucidating the role of NaV1.6 in peripheral sensory neurons

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