Chemical Synthesis and Characterization of an

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Chemical Synthesis and Characterization of an Equinatoxin II(1–85) Analogue John A. Karas, Marc-Antoine Sani and Frances Separovic * School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC 3010, Australia; [email protected] (J.A.K.); [email protected] (M.-A.S.) * Correspondence: [email protected]; Tel.: +61-3-9035-7539 Academic Editor: Margaret A. Brimble Received: 10 March 2017; Accepted: 29 March 2017; Published: 30 March 2017

Abstract: The chemical synthesis of an 85 residue analogue of the pore-forming protein, Equinatoxin II (EqtII), was achieved. Peptide precursors with over 40 residues were assembled by solid phase synthesis. The EqtII(1–46) fragment was modified to the reactive C-terminal thioester and native chemical ligation was performed with the A47C mutated EqtII(47–85) peptide to form the EqtII(1–85) analogue. Circular dichroism spectroscopy indicated that the N-terminal domain of EqtII(1–46) and EqtII(1–85) maintains predominantly an α-helical structure in solution and also in the presence of lipid micelles. This demonstrates the feasibility of assembling the full 179 residue protein EqtII via chemical means. Site-specific isotopic labels could be incorporated for structural studies in membranes by solid-state NMR spectroscopy. Keywords: native chemical ligation; solid-phase peptide synthesis; selectively labelled proteins; membrane protein structure; solid-state NMR

1. Introduction Equinatoxin II (EqtII), from the sea anemone Actinia equina is a 179 residue pore-forming toxin (PFT) and member of the actinoporin family [1,2]. According to nuclear magnetic resonance (NMR) and X-ray crystallographic data, it consists of a β-sheet core with a flexible α-helical N-terminal domain [3]. The mechanism of pore formation is well understood and proceeds via a number of distinct stages. Firstly, EqtII monomers bind to cell surfaces via the interaction of aromatic residues with sphingomyelin (SM) rich membranes [4]. The N-terminal α-helix then inserts into the membrane, followed by oligomerization and cell permeabilization [5,6]. There is evidence that the size of the pores that are formed are heterogeneous, as determined by conductance measurements. It is estimated that average pore consists of 3–4 monomers although higher oligomers are also possible [7]. However, the structure of the pore is not well characterized, such as the nature of the oligomeric interface between monomers and how lipids are involved in its architecture [5]. Therefore, alternative techniques need to be employed to aid in elucidating the mechanism of action of EqtII in SM rich membranes. Solid-state NMR spectroscopy is a powerful tool for observing the molecular dynamics of proteins in membranes [8,9]. This technique is often enhanced by incorporating “NMR-active” nuclei such as carbon-13 and nitrogen-15 into a biologically expressed product [4], which significantly enhances the signal and simplifies interpretation of the spectra. This is typically achieved by spiking the protein expression media with isotopically enriched amino acids or precursor molecules such as glucose. Although this global labelling strategy is often effective, it is not optimal when analysis of a specific protein domain is desired. In these situations, site-specific labelling of key residues can be more advantageous [10]. Our long-term goal is to chemically synthesize specifically labelled carbon-13 and nitrogen-15 labelled EqtII analogues for 2D NMR experiments in model membranes [11,12]. This will allow for the incorporation of isotopically enriched amino acids at peripheral residues to detect Molecules 2017, 22, 559; doi:10.3390/molecules22040559

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intermolecular contacts between monomers. However, the chemical synthesis of ~20 kDa proteins is extremely Therefore, the scope ofenriched this work willacids involve the designresidues and assembly allow challenging. for the incorporation of isotopically amino at peripheral to detectof an Molecules 2017, 22, 559 2 of 7 intermolecular contacts between monomers. However, the chemical synthesis of ~20 kDa proteins is EqtII(1–85) analogue from two synthetic peptide fragments, to validate our strategy. extremely challenging. Therefore, the scope of this work will involve the design and assembly of an The of proteins viaofexpressed protein ligation (EPL) native chemical allowsynthesis for the incorporation isotopically enriched amino acids[13] at or peripheral residues ligation to detect(NCL) EqtII(1–85) analogue from two[14,15] synthetic peptide fragments, to validatewith our strategy. are well-established techniques and more recently, proteins 166, 228 and 312 residues intermolecular contacts between monomers. However, the chemical synthesis of ~20 kDa proteins is The synthesis of proteins via expressed protein ligation (EPL) [13] or native chemical ligation have extremely been assembled using NCL [16–18]. In brief, peptide possesses a C-terminal thioester challenging. Therefore, the scope of thisawork willthat involve the design and assembly of an can (NCL) are well-established techniques [14,15] and more recently, proteins with 166, 228 and 312 residues analogue fromthat twocontains synthetican peptide fragments, to validate oura strategy. react EqtII(1–85) with a second peptide N-terminal cysteine to form native amide bond via thiol have been assembled using NCL [16–18]. In brief, a peptide that possesses a C-terminal thioester can The synthesis of proteins via expressed protein ligation (EPL) [13] or nativeNCL chemical ligation exchange reports havetoshown canbond be achieved react and withintramolecular a second peptiderearrangement. that contains anRecent N-terminal cysteine form athat native amide via (NCL) are well-established techniques [14,15] and more recently, proteins with 166, 228 and 312 residues via 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis (Fmoc SPPS) by using a C-terminal thiol exchange and intramolecular rearrangement. Recent reports have shown that NCL can be achieved have been assembled using NCL [16–18]. In brief, a peptide that possesses a C-terminal thioester can hydrazide as a thioester surrogate solid-phase [19,20]. There have also been advances peptide production, via 9-fluorenylmethoxycarbonyl peptide synthesis (Fmoc SPPS) byinusing a C-terminal react with a second peptide that contains an N-terminal cysteine to form a native amide bond via such as microwave-assisted Fmoc SPPS [21].There Thishave technology the assembly of longer hydrazide as a thioester surrogate [19,20]. also beenenables advances in efficient peptide production, such thiol exchange and intramolecular rearrangement. Recent reports have shown that NCL can be achieved as microwave-assisted Fmoc SPPS [21]. Thisoftechnology enables the efficient assembly EqtII of longer peptides [22–24], which minimizes the number steps required for NCL. Unfortunately, does not via 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis (Fmoc SPPS) by using a C-terminal peptides [22–24], which minimizes thefor number of steps requiredbut forthis NCL. Unfortunately, EqtII does in contain any cysteines that are necessary thioester formation, residue can be incorporated hydrazide as a thioester surrogate [19,20]. There have also been advances in peptide production, such not contain anyas cysteines that are for thioester formation, but reaction. this residue can be alanine incorporated strategic positions a surrogate fornecessary alanine to facilitate theenables ligation residues as microwave-assisted Fmoc SPPS [21]. This technology the efficient Native assembly of longer in strategic positions as a surrogate for alanine to facilitate the ligation reaction. Native alanine residues are then regenerated via radical-initiated homolysis of the sulfurfor atom [25]. Using this strategy would peptides [22–24], which minimizes the number of steps required NCL. Unfortunately, EqtII does are then regenerated via radical-initiated homolysis of the sulfur atom [25]. Using this strategy would not contain any cysteines that are necessary for thioester formation, but this residue can be incorporated make it feasible to produce EqtII from four fragments that range from 39 to 52 residues (Figure 1). make it feasible to produce EqtII from four fragments that range from 39 to 52 residues (Figure 1). in strategic positions as a surrogate for alanine to facilitate the ligation reaction. Native alanine residues are then regenerated via radical-initiated homolysis of the sulfur atom [25]. Using this strategy would make it feasible to produce EqtII from four fragments that range from 39 to 52 residues (Figure 1).

Figure 1. (a) Amino acid sequence of Equinatoxin II (EqtII). The alanine residues to be incorporated

Figure 1. (a) Amino acid sequence of Equinatoxin II (EqtII). The alanine residues to be incorporated as cysteine are in bold; (b) Proposed strategy for the chemical assembly of native EqtII. Note that the as cysteine are in bold; (b) Proposed strategy for the chemical assembly of native EqtII. Note that the scope of the work reported here involves the native chemical ligation (NCL) of the EqtII(1–46) and 1.work (a) Amino acid sequence of Equinatoxin II (EqtII). Theligation alanine residues to the be incorporated scopeFigure of the here involves the native chemical (NCL) EqtII of EqtII(1–46) EqtII(47–85) to reported give the EqtII(1–85) analogue. The crystal structure of full-length (PDB: 1IAZ) is and as cysteine are inthe bold; (b) Proposed strategy The for the chemical assembly of native EqtII. Note that 1IAZ) the EqtII(47–85) to give EqtII(1–85) analogue. crystal structure of full-length EqtII (PDB: is shown on the right. scope of the work reported here involves the native chemical ligation (NCL) of the EqtII(1–46) and shown on the right. EqtII(47–85) to give the EqtII(1–85) analogue. The crystal structure of full-length EqtII (PDB: 1IAZ) is 2. Results and Discussion shown on the right.

2. ResultsFor andthis Discussion study, EqtII fragments 1–46 and 47–85 were isolated as C-terminal hydrazides with the latter possessing an A47C mutation. The 3-Methylpentyl protection was used for the β-carboxylate

2. Results and Discussion For this study, EqtII fragments 1–46 and 47–85 were isolated as C-terminal hydrazides with the of aspartate residues instead of tert–butyl to minimize aspartimide formation and its associated side For this study, EqtIImutation. fragments The 1–46 3-Methylpentyl and 47–85 were isolated as C-terminal hydrazides with the latter possessing an A47C protection was used for the β-carboxylate products [26]. Purified 1–46 was oxidized to the acyl azide which facilitated the formation of the latter possessing an A47C mutation. The 3-Methylpentyl protection was used for the β-carboxylate of aspartate instead of tert–butyl to minimize and its associated reactive residues 4-mercaptophenylacetic acid (MPAA) thioester aspartimide followed by formation a second purification [19]. of aspartate[26]. residues instead of tert–butyl to minimize aspartimide formation and its associated side of side products Purified 1–46 was oxidized to the acyl azide which facilitated formation An equimolar amount of purified 47–85 was added to the reaction mixture and thethe ligation was products4-mercaptophenylacetic [26]. Purified 1–46 was oxidized to the acyl azide which facilitated the formation of the the reactive performed at neutral pH (Figure 2).acid (MPAA) thioester followed by a second purification [19]. reactive 4-mercaptophenylacetic acid (MPAA) thioester followed by a second purification [19]. An equimolar amount of purified 47–85 was added to the reaction mixture and the ligation was An equimolar amount of purified 47–85 was added to the reaction mixture and the ligation was performed at neutral pH (Figure 2). performed at neutral pH (Figure 2).

Figure 2. Reaction scheme for the formation of the EqtII(1–85) analogue. (a): pH 7, 20 °C, 18 h; (b): reversed-phase high performance liquid chromatography (RP-HPLC). Figure 2. Reaction scheme for the formation of the EqtII(1–85) analogue. (a): pH 7, 20 °C, 18 h; Figure 2. Reaction scheme for the formation of the EqtII(1–85) analogue. (a): pH 7, 20 ◦ C, 18 h; (b): reversed-phase high performance liquid chromatography (RP-HPLC). (b): reversed-phase high performance liquid chromatography (RP-HPLC).

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The reaction reaction was wasmonitored monitoredbybyreversed-phase reversed-phase high performance liquid chromatography The high performance liquid chromatography (RP(RP-HPLC) (Figure 3a) and the product was characterized via analytical RP-HPLC and electro-spray HPLC) (Figure 3a) and the product was characterized via analytical RP-HPLC and electro-spray ionization mass mass spectrometry spectrometry (ESI-MS) (ESI-MS) (Figure (Figure 3b,c, 3b,c, respectively). respectively). Once Once the the reaction reaction was was complete, complete, ionization the EqtII(1–85) C-terminal hydrazide analogue was isolated as the A47C disulfide-linked MPAA the EqtII(1–85) C-terminal hydrazide analogue was isolated as the A47C disulfide-linked MPAA adduct (which can be reductively cleaved and desulfurized in a subsequent step) in a yield of adduct (which can be reductively cleaved and desulfurized in a subsequent step) in a yield of 12%. 12%.

Figure 3. (a) NCL reaction after 4 h; (b) analytical RP-HPLC of the purified EqtII(1–85) analogue and Figure 3. (a) NCL reaction after 4 h; (b) analytical RP-HPLC of the purified EqtII(1–85) analogue (c) ESI-MS of the purified EqtII(1–85) analogue (inset: Deconvoluted spectrum; molecular mass and (c) ESI-MS of the purified EqtII(1–85) analogue (inset: Deconvoluted spectrum; molecular mass (calc.) = 9091.4 Da). Please note that chromatograms (a,b) were analyzed using different HPLC (calc.) = 9091.4 Da). Please note that chromatograms (a,b) were analyzed using different HPLC instruments, which accounts for the small difference in the retention time of the EqtII(1–85) analogue. instruments, which accounts for the small difference in the retention time of the EqtII(1–85) analogue.

All three fragments were analyzed via circular dichroism (CD) spectroscopy in phosphate buffer fragments were analyzed via circular dichroism (CD) spectroscopy in phosphate buffer at at pHAll 7.2,three to determine whether these synthetic analogues possess a secondary structure similar to native pH 7.2, to determine whether these synthetic analogues possess a secondary structure similar to native EqtII. The CD lineshape of EqtII(1–46) in solution showed typical features of an α-helical structure EqtII.minima The CD at lineshape EqtII(1–46) in solution showed typical features of an structuresince with with 222 nm of and 209 nm and a maximum at ca. 190 nm (Figure 4a).α-helical This is expected minima at 222 nm and 209 nm and a maximum at ca. 190 nm (Figure 4a). This is expected since the the N-terminal domain can readily translocate and, therefore, does not require any structural N-terminal domain can readily translocate and, therefore, doesCD not studies require any structural constraints to constraints to maintain its native conformation. NMR and of the N-terminus of EqtII maintain its native conformation. NMR and CD studies of the N-terminus of EqtII report an α-helical report an α-helical structure [27–29]. In the presence of either dodecylphosphocholine (DPC) or sodium structuresulfate [27–29]. In the presence either dodecylphosphocholine (DPC)increased or sodium dodecyl dodecyl (SDS) micelles, the of intensity of the minima and maximum (Figure 4a),sulfate which (SDS) micelles, the intensity of the minima and maximum increased (Figure 4a), which indicates indicates a greater helicity of the peptides. By contrast, EqtII(47–85) in solution had a CD lineshape a greater helicity of theatpeptides. contrast, EqtII(47–85) had(Figure a CD 4b). lineshape with with a single minimum ca 195 nmBy that is typical of random in coilsolution structures There is no a single minimum at ca 195 nm that is typical of random coil structures (Figure 4b). There is no β-sheet formation exclusively within this fragment in native EqtII according to X-ray crystallographic β-sheet exclusively within this fragment in native EqtII according to X-ray data [3],formation so this peptide, untethered from the native protein, would be unlikely tocrystallographic adopt a highly data [3], so this peptide, untethered from the native protein, would be unlikely to adopt a highly ordered conformation. ordered conformation. In the presence of DPC or SDS micelles, however, the CD spectra showed a reduction and a red In the or SDS micelles, however, the minimum CD spectra reduction which and a shift of the presence minimumofatDPC 195 nm to 209 nm, while a second atshowed ~222 nma appeared, red shift of the minimum at 195 nm to 209 nm, while a second minimum at ~222 nm appeared, indicates a significant increase in α-helical content. EqtII(1–85) was insoluble in the phosphate buffer which indicatesaaCD significant increase in recorded. α-helical content. EqtII(1–85) was insoluble in the phosphate and, therefore, spectrum was not The fragment was soluble in the presence of DPC buffer and, therefore, a CD spectrum was not recorded. The fragment was soluble in presence of and SDS micelles (Figure 4c) and appeared to possess the expected α-helicity in both the systems, given DPC and SDS micelles (Figure 4c) and appeared to possess the expected α-helicity in both systems, that it contains the N-terminal domain. In short, all CD spectroscopic data supports the notion that given chemically that it contains the N-terminal In short, CD spectroscopic datastructure supportsin the notion these synthesized peptidedomain. fragments adopt all a predictable secondary solution that these chemically synthesized peptide fragments adopt a predictable secondary structure in solution (or lack thereof). EqtII(1–46) and EqtII(1–85) also appear to exhibit behavior similar to the native (or lack in thereof). EqtII(1–46) and EqtII(1–85) also appear to exhibit behavior to the native protein 1H solution protein the presence of membrane systems. The NMR spectrasimilar of EqtII(1–46), EqtII(47–85) 1 H solution NMR spectra of EqtII(1–46), EqtII(47–85) and in the presence of membrane systems. The 1 and EqtII(1–85) in DPC micelles exhibited H chemical shift dispersions and sharp resonances, 1 H chemical shift dispersions and sharp resonances, supporting EqtII(1–85) inhigh DPChelical micellescontents exhibited(See supporting Supplementary Material S1). Finally, these results with high helical contents (See Supplementary Material S1).beFinally, thesesynthesized results withand EqtII(1–85) indicate EqtII(1–85) indicate that full length EqtII(1–179) may chemically likely adopt the that full length EqtII(1–179) may be chemically synthesized and likely adopt the correct conformation. correct conformation.

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Figure 4. Circular dichroism (CD) spectroscopy of the: (a) EqtII(1–46) fragment; (b) EqtII(47–85) A47C Figure 4. Circular dichroism (CD) spectroscopy of the: (a) EqtII(1–46) fragment; (b) EqtII(47–85) A47C fragment; (c) EqtII(1–85) fragment (as the 4-mercaptophenylacetic acid (MPAA) adduct). ~40 μM fragment; (c) EqtII(1–85) fragment (as the 4-mercaptophenylacetic acid (MPAA) adduct). ~40 µM peptide/protein in buffer, 15 mM dodecylphosphocholine (DPC) or sodium dodecyl sulfate (SDS) peptide/protein in buffer, 15 mM dodecylphosphocholine (DPC) or sodium dodecyl sulfate (SDS) micelles, pH 7.2, 25 °C. micelles, pH 7.2, 25 ◦ C.

3. Experimental Section 3. Experimental Section 3.1. Materials Materials 3.1. Peptidesynthesis synthesiswas was performed performed on on aa CEM CEM Liberty Liberty microwave microwave peptide peptide synthesizer synthesizer (CEM (CEM Corp., Corp., Peptide Matthews, NC, USA). Standard Fmoc amino acids and Oxyma Pure were also purchased from CEM. Matthews, NC, USA). Standard Fmoc amino acids and Oxyma Pure were also purchased from CEM. Fmoc-Asp(OMpe)-OH, Boc-Gly-OH and 2-Chlorotrityl chloride resin (sub: 1.0 mmol/g) were Fmoc-Asp(OMpe)-OH, Boc-Gly-OH and 2-Chlorotrityl chloride resin (sub: 1.0 mmol/g) were obtained obtained from GL(Shanghai, Biochem (Shanghai, China). Diisopropylcarbodiimide, hydrochloride from GL Biochem China). Diisopropylcarbodiimide, guanidine guanidine hydrochloride (Gn.HCl), (Gn.HCl), diethyl ether, acetonitrile, dimethylformamide (DMF) and dichloromethane were sourced diethyl ether, acetonitrile, dimethylformamide (DMF) and dichloromethane were sourced from from Merck (Darmstadt, Germany). Sodium nitrite, sodium hydrogen phosphate (Na 2HPO4), hydrazine, Merck (Darmstadt, Germany). Sodium nitrite, sodium hydrogen phosphate (Na2 HPO4 ), hydrazine, diisopropylethylamine (DIEA), (DIEA), piperidine, piperidine, trifluoroacetic trifluoroacetic acid acid (TFA), (TFA), triisopropylsilane triisopropylsilane (TIPS), (TIPS), diisopropylethylamine 3,6-dioxa-1,8-octanedithiol (DODT) and MPAA were obtained from Sigma (Sydney, Australia). Dodecyl 3,6-dioxa-1,8-octanedithiol (DODT) and MPAA were obtained from Sigma (Sydney, Australia). Dodecyl phosphocholine (DPC) (DPC) and Sigma (Australia). phosphocholine and sodium sodiumdodecyl dodecylsulfate sulfate(SDS) (SDS)were wereobtained obtainedfrom from Sigma (Australia). 3.2. Peptide Peptide Synthesis Synthesis 3.2. Thepeptide peptidefragments fragments were wereisolated isolatedas asC-terminal C-terminalhydrazides hydrazidesusing usingstandard standardmicrowave-assisted microwave-assisted The Fmoc-SPPSprotocols. protocols. Briefly, Briefly,2-chlorotrityl 2-chlorotritylchloride chlorideresin resin(0.3 (0.3mmol) mmol)was wastreated treatedwith withexcess excesshydrazine hydrazine Fmoc-SPPS and 55 eq. eq. of of DIEA, DIEA, followed followed by by peptide peptide assembly. assembly. The The first first amino amino acid acid was was spiked spiked with with 22eq. eq. of of and Boc-Gly-OHtotoreduce reduce resin loading. All amino were coupled 90 °Cfor except for Boc-Gly-OH thethe resin loading. All amino acidsacids were coupled once atonce 90 ◦ Catexcept arginine arginine which wastwice coupled andwhich histidine was at 50 °C. Deprotection was which was coupled and twice histidine waswhich coupled at coupled 50 ◦ C. Deprotection was performed ◦ performed with 20% piperidine in DMF and 0.1 M Oxyma at75 either or ambient temperature (when with 20% piperidine in DMF and 0.1 M Oxyma at either C 75 or °C ambient temperature (when an an aspartate residue was present). Cleavage and global deprotectionwas wasperformed performedwith withaacocktail cocktail of of aspartate residue was present). Cleavage and global deprotection TIPS/H 2 O/DODT/TFA (2%/2%/1%/95%), followed by ether precipitation and RP-HPLC purification. TIPS/H2 O/DODT/TFA (2%/2%/1%/95%), followed by ether precipitation and RP-HPLC purification. The C-terminus C-terminus of of the the N-terminal N-terminal fragment fragment was was converted converted to the the MPAA MPAA thioester thioester via via aa previously previously The reportedmethod method[19], [19],followed followedby byaasecond secondpurification. purification. reported 3.3. 3.3. Purification Purification and and Analysis Analysis HPLC analysisand andpurification purification were performed an Agilent system CA, HPLC analysis were performed on on an Agilent 11001100 system (Santa(Santa Clara,Clara, CA, USA). USA). Mobile phases: (1) buffer A = 0.1% TFA in water; buffer B = 0.1% TFA in acetonitrile. Purification Mobile phases: (1) buffer A = 0.1% TFA in water; buffer B = 0.1% TFA in acetonitrile. Purification was was carried outa on a Phenomenex Kinetex µm XB-C18, 150 mm mm × 21.2 mmpacked AXIA column. packed carried out on Phenomenex Kinetex 5 μm 5XB-C18, 100 Å,100 150Å, mm × 21.2 AXIA Typical gradient: 10–70% buffer B over 60 min with a 5 mL/min flow rate and detection at 230 nm. Analysis was performed on a Phenomenex 5 μm C18, 100 Å, 150 mm × 4.6 mm column. Typical gradient:

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column. Typical gradient: 10–70% buffer B over 60 min with a 5 mL/min flow rate and detection at 230 nm. Analysis was performed on a Phenomenex 5 µm C18, 100 Å, 150 mm × 4.6 mm column. Typical gradient: 0–80% buffer B over 40 min with a 1 mL/min flow rate and detection at 220 nm. Mass spectrometry was performed on an Agilent 6510 ESI-TOF instrument. All peptides were lyophilized on a Virtis freeze dryer. 3.4. Native Chemical Ligation EqtII(1–46) thioester (3.0 mg, 1 eq.) and EqtII(47–85) A47C (2.7 mg, 1 eq.) were dissolved in a solution of 6 M Gn.HCl and 0.2 M Na2 HPO4 at pH 7 with MPAA (4.5 mg, 50 eq.). The reaction was stirred for 18 h at 20 ◦ C, followed by purification and lyophilization. An amount of 0.6 mg of purified material was recovered, indicating a yield of 12%. Refer to Figure 3b,c for analytical data. 3.5. CD Spectroscopy Detergent-free peptide solutions were dissolved in 10 mM phosphate buffer solution (NaCl 1 mM, pH 7.4) to a final concentration of ~40 µM. Appropriate aliquot of 200 mM DPC or SDS micellar stock solutions were added to the peptides to reach a final detergent and peptide concentrations of 15 mM and 40 µM, respectively. CD spectra were acquired on a Chirascan spectropolarimeter (Applied Photophysics Ltd., Leatherhead, UK) between 190 and 260 nm using a 1 mm pathlength quartz cell (Starna, Hainault, UK). Spectra were acquired with 1 nm data intervals, 0.5 s integration time and three scans accumulation. The signal was recorded as milli-degrees at 25 ◦ C. 4. Conclusions In conclusion, the chemical synthesis of an 85 residue EqtII analogue was achieved. By employing microwave-assisted Fmoc SPPS, peptide precursors with over 40 residues were assembled efficiently. After modification of the EqtII(1–46) fragment to the reactive C-terminal thioester, NCL was performed with the A47C mutated EqtII(47–85) peptide. The 1–85 analogue was formed without the formation of any significant side products and isolated via RP-HPLC at high purity. CD spectroscopy indicated that the N-terminal domain of EqtII(1–46) and EqtII(1–85) maintains predominantly an α-helical structure in solution and also in the presence of DPC and SDS micelles. Finally, this work demonstrates the feasibility of assembling the ~20 kDa protein toxin EqtII via chemical means. Once all ligation and desulfurization reactions are optimized, site-specific isotopic labels could then be incorporated to aid in the elucidation of its mechanism of action with membranes via solid-state NMR spectroscopy. Supplementary Materials: The following are available online: NMR spectra of the peptides. Acknowledgments: The authors acknowledge finding from the Australian Research Council for DP140102127 grant awarded to F.S. Author Contributions: J.A.K., F.S. and M.-A.S. conceived and designed the experiments; J.A.K. and M.-A.S. performed the experiments and analyzed the data; J.A.K., M.-A.S. and F.S. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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