Synthesis, Structural Characterization, and Bioactivity of the Stable

Article pubs.acs.org/jnp

Synthesis, Structural Characterization, and Bioactivity of the Stable Peptide RCB‑1 from Ricinus communis Delgerbat Boldbaatar,†,‡ Sunithi Gunasekera,† Hesham R. El-Seedi,†,§ and Ulf Göransson*,† †

Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Box 574, SE-751 23 Uppsala, Sweden ‡ School of Engineering and Applied Sciences, National University of Mongolia, Ulaanbaatar-46, Mongolia § Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ABSTRACT: The Ricinus communis biomarker peptides RCB-1 to -3 comprise homologous sequences of 19 (RCB-1) or 18 (RCB-2 and -3) amino acid residues. They all include four cysteine moieties, which form two disulfide bonds. However, neither the 3D structure nor the biological activity of any of these peptides is known. The synthesis of RCB-1, using microwave-assisted, Fmocbased solid-phase peptide synthesis, and a method for its oxidative folding are reported. The tertiary structure of RCB-1, subsequently established using solution-state NMR, reveals a twisted loop fold with antiparallel β-sheets reinforced by the two disulfide bonds. Moreover, RCB-1 was tested for antibacterial, antifungal, and cytotoxic activity, as well as in a serum stability assay, in which it proved to be remarkably stable.

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RESULTS AND DISCUSSION Isolation and Identification of RCB-1. Extraction of R. communis was performed by using dilute acid, HCOOH in H2O, mixed with CH3CN. The extract was subjected to solidphase extraction on a C18 column. After freeze-drying, the resulting fraction was dissolved and analyzed using LC-MS. The analysis showed triply and doubly charged ion pairs at m/z 690.03+/1034.52+, m/z 660.63+/990.92+, and m/z 654.73+/ 982.02+ (Figure 1A). This finding matched the study by Ovenden and co-workers in which three novel small peptides, RCB-1 to -3, were reported as biomarkers of the seed extract of R. communis.5 The calculated masses ([M + H]+) of RCB-1 to -3 were 2066.90, 1979.78, and 1961.84 Da, respectively. The sequence that corresponded in mass with RCB-1 was selected for synthesis to get a sufficient amount of peptide for NMR studies and bioassays. RCB-1 consists of 19 amino acids and comprises two disulfide bonds (Figure 1B). Peptide Synthesis and Oxidative Folding. RCB-1 was synthesized on a Ser-preloaded Tentagel resin by Fmoc solidphase peptide synthesis (SPPS) with the assistance of microwave heating during deprotection and coupling steps. The yield was ∼50% based on the increase in weight of the resin. From 102.2 mg of cleaved peptide, 16.9 mg of reduced RCB-1 was obtained after purification on RP-HPLC. The peptide was then subjected to oxidative folding. Ten different folding conditions (Table 1), varying in concentrations of redox agents and cosolvents, were tested to determine the best folding protocol for RCB-1. Retention times (analytical RP-HPLC) and masses of RCB-1 derived

lant peptides constitute a growing class of natural products, including several families of biologically active peptides such as cyclotides, orbitides, trypsin inhibitors, and plant defensins. Some of those peptides are being considered as possible drug leads or show promise as a biotechnological tool.1,2 In medicine, peptides find increasing use as biomarkers of physiological and pathological conditions. In plants, such peptide biomarkers could be used to study plant biology and phylogeny or to identify species. Ricinus communis (L.) (Euphorbiaceae), the castor oil plant, is well known for its content of the protein ricin, which is one of the most toxic natural poisons from the plant kingdom.3 There have been many case reports of ricin poisoning, as it is found in high concentrations in the plant and is easily extractable. Owing to this latter property ricin is also a potential warfare agent.4 Interest in the toxic features of R. communis seeds and in their use for bioterrorism has led to an overall focus on a search for biomarkers that identify the noxious agent in forensic samples and the plant itself. For example, the alkaloid ricinine has been used as a biomarker in the case of castor bean poisoning,4 and three unique peptides designated as R. communis biomarkers (RCB) 1−3 were recently identified in the seeds of geographically different cultivars.5 In the current work, the RCB peptides from R. communis, triggered by their unique peptide sequence and suggested roles as antimicrobial agents, are revisited. The synthesis, oxidative folding, and the 3D structure of RCB-1 and antibacterial, antifungal, and cytotoxic activities are reported. Furthermore, the stability of RCB-1 in human serum, and thus the potential of this peptide as a biomarker for castor bean intoxication, is demonstrated. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 22, 2015

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DOI: 10.1021/acs.jnatprod.5b00463 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. (A) LC-MS chromatogram of the seed extract of R. communis. Triply charged, protonated, molecular ions of RCB-1, RCB2, and RCB-3 were detected at m/z 690.0, 660.6, and 654.7, respectively. (B) Amino acid sequences and disulfide bond connectivities of RCB-1 to -3 biomarkers of R. communis according to Ovenden and co-workers.5 Figure 2. (A) HPLC profile of the folding reaction condition #8 in which the oxidized methionine (+16 Da, 1042.62+) was observed. Methionine-oxidized (folded, partially folded, or misfolded) conformations are marked with arrows. The asterisked (*) peak corresponds to natively folded RCB-1 (1034.52+) without methionine oxidation. (B) HPLC profile of folded RCB-1. The correctly folded peptide is annotated*. Other peaks correspond to misfolded and partially folded peptides.

Table 1. Oxidative Folding Trials trial #

peptide (1 mg/mL), μL

0.1 M NH4HCO3, μL

1 2 3 4 5 6 7 8 9 10

20 20 20 20 20 20 20 20 20 20

180 90 160 80 140 70 40 60 20 40

iPrOH, μL

GSG (20 mM), μL

GSSG (4 mM), μL

20 20 20 20

20 20

20 20

20 20

90 80 70 140 120 120 100

from the different folding mixtures were compared to that of native RCB-1 as isolated from the seeds. It proved difficult to obtain native RCB-1 during the initial attempts at oxidative folding, due to oxidation of the single methionine moiety, resulting in a mass increase of +16 Da (Figure 2A). However, the highest yield of successfully folded peptide was obtained in condition #8, which contained 60% i-PrOH (v/v) and 0.1 M NH4HCO3. The kinetics of folding in that buffer was then monitored over time (2 min, 1 h, 2 h, 4 h, 8 h, and 24 h) to find the best interval for folding with a minimal loss of peptide to methionine oxidation. Oxidative folding of RCB-1 proved to be fast, and a reaction time of 2 min was found to be the most appropriate condition to avoid methionine oxidation. At that time point, only small amounts of oxidized methionine and misfolded peptides still are detected (Figure 2B). Subsequently, reduced RCB-1 peptide was folded in a large-scale reaction using condition #8 over 2 min. The final product was purified by RP-HPLC and analyzed by QTOF-MS. The analytical HPLC comparison of synthetic and folded RCB-1 to native peptide isolated from seeds is shown in Figure 3. NMR Spectroscopy and Resonance Assignments. For determining the structure of RCB-1, 1H NMR spectra were recorded at 800 MHz using ∼1 mM of peptide in 90% H2O/ 10% D2O (600 μL) at pH 5. The spectra showed well-dispersed resonances, confirming that the peptide was correctly folded and highly structured in solution. Complete sequence-specific resonance assignments were achieved by analyzing DQF-

Figure 3. Synthetically folded RCB-1 (bottom) elutes at the same time as the native peptide (top). The peak corresponding to RCB-1 is highlighted.

COSY, TOCSY, and NOESY spectra obtained at 290 and 298 K. Initially, individual spin systems were identified in the NHHα fingerprint region of the TOCSY spectrum (Figure 4A). The sequential resonance assignments were achieved by linking individual spin systems via sequential dαN(i,i+1) connectivities in the fingerprint region of the NOESY spectrum (Figure 4B). Analysis of the secondary Hα chemical shifts (Hα chemical shift for a specific residue compared to that of the corresponding residue in random coil conformation) suggested that RCB-1 possesses secondary structure elements, namely, two antiparallel β-strands (Figure 5). Resonance assignments for Cα, Cβ, Cγ, and N were also performed using 13C and 15N HSQC spectra. The cis−trans conformations of proline residues were determined by observing NOE cross-peaks and examining the difference in chemical shifts between Cβ and Cγ in 13C HSQC spectra.6 As highlighted in Figure 6, strong NOE peaks of B



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Figure 4. (A) NH-Hα fingerprint region of the TOCSY spectrum of RCB-1 showing intraresidue connectivities. (B) Sequential dαN(i,i+1) connectivities in the NH-Hα fingerprint region of the NOESY spectrum.

Figure 5. Secondary Hα chemical shift analysis of RCB-1. The secondary Hα shifts were calculated by subtracting the random coil shifts from the experimental Hα shifts. Stretches of positive values indicate the presence of β-strands.

dαδ(7,8) and dαδ(9,10) were observed in the NOESY spectrum, highlighting the presence of a trans conformation preceding Pro8 and Pro10. On the contrary, prominent NOEs for dαα(10,11) suggested the presence of a cis-proline bond preceding Pro11. Given that the Δβγ of 13Cβ and 13Cγ for a proline is 9.15 ppm for a proline suggests a cis conformation.6 In the 13C HSQC spectrum, Δβγ of 13Cβ and 13 γ C for Pro8 and Pro10 were 4.7 and 3.15 ppm, respectively, confirming the trans conformation. Pro11 gave an ambiguous Δβγ of 7.8 ppm, but a 13Cβ chemical shift of 32.6 ppm and

Figure 6. Hα−Hα region of the NOESY spectrum. The dαα(i−1,i) and dαδ(i−1,i) connectivities of prolines.

strong dαα(10,11) NOE in the NOESY spectrum confirmed it as a cis-proline. Structure Determination of RCB-1. The tertiary structure of RCB-1 was calculated by CYANA 3.0 using distant constraints derived from NOEs, dihedral angles, and Hbonds. Two disulfide connectivities, between Cys3−Cys14 and Cys4−Cys18, previously determined by Ovenden and coC



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Figure 7. Three-dimensional structure of RCB-1. (A) Superimposition of the 15 lowest energy structures. (B) Ribbon structure of RCB-1 with the lowest energy. The disulfide bonds and β-strands are shown in yellow and cyan, respectively. (C) Molecular surface representation of RCB-1. Hydrophobic residues are in white, polar residues in green, and charged residues in red color.

workers using MS-MS were used in the structure calculation. The results confirmed that these two disulfide bond connectivities are the most energetically favorable, as alternative connectivities led to a higher target function and multiple distant/angle violations. Hydrogen−deuterium exchange followed by TOCSY experiments for RCB-1 indicated several slowly exchanging amide protons. In particular, the backbone amide protons of residues 4, 5, 6, 7, 9, 12, 13, 15, 16, and 18 were still detectable after 24 h, indicative of strong hydrogen bonding. Hydrogen bonds between Cys18 (O)−Arg2 (HN), Lys16 (O)−Cys4 (HN), Lys16 (O)−Leu5 (HN), Ala13 (O)− Met7 (HN), and Leu5 (O)−Val15 (HN) were deduced from analysis of preliminary structures, and slow exchange data were included in the structure calculations. An ensemble of the 15 lowest energy structures was selected from a final set of 50 to represent the structure of RCB-1 in solution (Figure 7). The structure had no distance violations greater than 0.2 Å and no dihedral angle violations greater than 3°. The root-mean-square-deviations (RMSDs) of backbone and heavy atoms in residues 1−19 were 0.15 and 0.69 Å, respectively. Validation of the structural statistics using the Richardson Lab’s MolProbity4 server (http://molprobity. biochem.duke.edu) demonstrated that all the backbone dihedral angles are within the allowed regions of a Ramachandran plot.7 Detailed structural statistics are given in Table 2. An insight into the RCB-1 structure reveals that the peptide backbone adopts a twisted loop shaped conformation with antiparallel β-sheets that are reinforced by two disulfide bonds: Cys3−Cys14 and Cys4−Cys18. Bioactivity and Serum Stability. RCB-1 was suggested by Ovenden and co-workers to be a putative plant defensin peptide having a role in protecting seeds against pathogen invasions, based on sequence similarity with the antimicrobial peptides Ib-AMP 1 to 4.5 Encouraged by the hypothesis, we evaluated the antibacterial and antifungal activity of RCB-1. Different concentrations of RCB-1, ranging from 160 to 0.625 μM, were tested against human and plant pathogens. The results are shown in Table 3. The peptide was active, at a concentration of 27 μM, against human pathogen Pseudomonas aeruginosa. The results of an MIC assay showed that RCB-1, at concentrations up to 160 μM, was inactive against the human pathogens Esherichia coli, Staphylococcus aureus, and Candida albicans and against the phytopathogens Erwinia carotovora,

Table 2. NMR and Refinement Statistics for RCB-1 Peptide parameter

value

NMR Distance and Dihedral Constraints interproton distance restraints intraresidue, |i−j| = 0 sequential, |i−j| = 1 short-range, |i−j| ≤ 1 medium range, 1 < |i−j| < 5 long range, |i−j| ≥ 5 dihedral angle restraints hydrogen bond restraintsa disulfide bond restraints Structure Statistics CYANA target function (Å) CYANA residues in most favored Ramachandran region, % violations (mean and SD) distance constraints (>0.1 Å) dihedral angle constraints (>3°) stereochemical qualityb residues in most favored Ramachandran region, % Ramachandran outliers, % unfavorable side chain rotamers, % Clashscore, all atoms overall Molprobity score RMS deviations from mean coordinate structure (Å) backbone atoms all heavy atoms

272 65 93 158 41 73 23 5 4 0.68 ± 1.5 85.4 0.2/structure 0 100 ± 0 0±0 14.51 ± 4.9 6.69 ± 3.8 2.19 ± 0.24 0.15 ± 0.07 0.69 ± 0.08

a

Two restraints were used per hydrogen bond. bStereochemical quality was assessed using MolProbity. Clashscore is defined as the number of steric overlaps >0.4 Å per thousand atoms.

Erwinia amylovora, and Aspergillus f umigatus. Furthermore, a cytotoxicity assay indicated that RCB-1 was nontoxic to human lymphoma cells (U-937 GTB) up to a concentration of 50 μM. An investigation of the stability of RCB-1 in human serum at 37 °C found that >95% of the peptide remained in serum for up to 24 h, demonstrating a remarkable stability of RCB-1 and its resistance to enzyme degradation in serum (Figure 8). As such RCB-1 was found to be active against only Pseudomonas aeruginosa when it was tested against a set of common human and plant pathogens. Although RCB-1 may not be involved in the seed protection like defensin peptides, its remarkable stability in human serum for up to 24 h indicates its D



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economically valuable plants such as grapevine.13 In addition, proteomic profiling can also be correlated to biochemical and physiological changes in a plant to understand berry development, the ripening process, and underlying metabolic processes.14 RCB-1 could be released at a specific development stage in R. communis, and this may be economically important for specific products from this plant. Thus, it is possible that ascertaining the presence and concentration of RCB-1 at different stages of seed development will be useful. Subsequent association of RCB-1 with a key developmental or metabolic stage may open up possibilities for controlling seed development, which would also be economically valuable. To conclude, we have described the identification, synthesis, 3D structure, and bioactivity screening of RCB-1 in the current study. The function of this peptide, derived from the seeds of R. communis, in planta is yet unknown. RCB-1 was found to be remarkably stable in human serum. Further experiments are needed to determine if RCB-1 is present in ricin-containing extracts or to determine if RCB-1 would be useful for controlling seed development or for the identification of different varieties.

Table 3. Antibacterial and Antifungal Activity of RCB-1 Peptide

a

microorganism

EC50, μMa

Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Erwinia carotovora Erwinia amylovora Candida albicans Aspergillus f umigatus

>160 >160 27 ± 5.7 >160 >160 >160 >160

Mean ± SD of three replicates.

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EXPERIMENTAL SECTION

General Experimental Procedures. The NMR spectra were recorded at 800 MHz using a Bruker Avance HD III 800 MHz spectrometer equipped with a 5 mm TCI cryoprobe (Bruker, Billerica, MA, USA). LC-MS was performed in ESI positive ion mode using a Waters NanoAcquity UPLC connected to a Waters Micromass QTOF Micro MS (Waters, Milford, MA, USA). HPLC analysis was performed on an Ä kta Basic 10 HPLC system with a UV-900 detector (GE Healthcare, Sweden). The peptide was synthesized on a Liberty1 microwave peptide synthesizer (CEM, Matthews, NC, USA). Plant Materials. Seeds were collected from South Sinai, Egypt, in 2009 and identified by Prof. Loutfy Boulos (Department of Botany, Faculty of Science, Alexandria University, Egypt). A voucher specimen of R. communis, HRE 200, is deposited at the herbarium of the Department of Botany at the Faculty of Science, El-Menoufia University, Egypt. Peptide Extraction. Dried plant material (250 mg) were ground and subjected to extraction with 4 mL of 60% CH3CN in H2O, containing 0.1% HCOOH, for 2 h at room temperature. The supernatant was collected, and the extraction repeated with 4 mL of 30% CH3CN in H2O, containing 0.1% HCOOH, and finally with 4 mL of 0.1% HCOOH. The pooled supernatants were diluted with 0.1% HCOOH in H2O to a final concentration of 10% CH3CN. Subsequently, the diluted samples were loaded onto a 500 mg C18 SPE column (Isolute, Biotage, Sweden) and eluted first with solvent A (10% CH3CN/0.1% HCOOH) and then solvent B (60% CH3CN/ 0.1% HCOOH). The fractions eluted with solvents A and B were freeze-dried and dissolved in solvent A for LC-MS analysis. Peptide Synthesis. RCB-1 was synthesized on Tentagel R PHBSer (0.18 mmol/g) resin (Rapp Polymere, Germany) on a 0.1 mmol scale using a microwave-assisted Fmoc/2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) SPPS protocol. Fmoc-amino acids were Fmoc-Ala-OH, Fmoc-Arg(Pbf)OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, and Fmoc-Val-OH. Deprotection of Fmoc amino acids was carried out using 20% piperidine in DMF for 3 min at 70 °C. Coupling for standard amino acids, except cysteine, was done using 0.5 M HBTU and 2 M N,Ndiisopropylethylamine (DIPEA) for 5 min at 70 °C. Cysteines were coupled at 50 °C for 4 min. Details of the deprotection conditions and coupling steps have been described previously.15 After synthesis, the peptide was cleaved from the resin (690 mg) using TFA/TIPS/H2O (95.5:0.25:0.25, 90 min, room temperature), then filtered and flushed with N2 to a volume of 0.5 mL. Cold Et2O was added, and the precipitated peptide was collected by centrifugation. The precipitate

Figure 8. Stability of RCB-1 in human serum. Points represent the mean ± SD of three replicates.

potential value as a biomarker in forensic analysis to determine ricin exposure. Ricinine has been applied as a potential biomarker in similar analyses by way of its detection in victims’ urine samples.8 In one attempt to determine peptide biomarkers for ricin intoxication, five serum peptides (m/z 4982.49, 1333.25, 1537.86, 4285.05, and 2738.88) were identified in mice in the mass range 1000−10 000 Da.9 Screening serum or blood and possibly also urine samples for RCB-1 could also be one way to determine exposure. Comparisons of the RCB-1 amino acid sequence to sequence databases (e.g., NCBInr database using protein BLAST and tBLASTn, UniProtKB using protein BLAST) failed to identify any homologous peptides, nor did searches for 3D structural homologues in the Protein Data Bank (RCSB) and Molecular Modeling Database (MMDB) using VAST Search10 results in any hits. The 3D structure of Ib-AMP1, which was predicted to be a similar peptide, appears to be the peptide most closely related to RCB-1, as pointed out by Ovenden and co-workers.5 Although the pdb file containing the structure of Ib-AMP1 is lacking in the structural databases, making it impossible to superimpose it on RCB-1, it is clear that these two peptides have similar disulfide connectivities and share the fold of βturns in a twisted loop conformation.11 So, can these biomarkers be used for anything other than forensic sciences? Although we do not yet know the function of the peptide, its origin from an economically valuable plant, R. communis, is the source of products such as castor oil, ricinoleic acid, and glycerol ricinoleate, from which numerous industrial, medical, and cosmetic commodities are produced.12 Considering that protein-based tools for identifying plant varieties are developing rapidly, in parallel with the evolution of gene-based techniques, screening different varieties of R. communis for RCB peptides could be used for characterizing the quality of certain products derived from that variety. In fact, protein profiling has been useful for identifying different varieties of many E



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mass at the doubly charged ion mass of 1034.52+. The control samples consisted of RCB-1 peptide diluted in phosphate-buffered saline, instead of serum, and were analyzed as described above. Antibacterial and Antifungal Assays. The activities of RCB-1 were evaluated using a minimum inhibitory concentration (MIC) assay against Staphylococcus aureus (ATCC29213), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Candida albicans (ATCC90028), Aspergillus f umigatus, Erwinia carotovora, and Erwinia amylovora in 96-well plates.21 In short, bacteria were grown overnight (3% TSB) at 37 °C, except E. carotovora and E. amylovora, which were grown at 27 °C, washed twice with Tris buffer (10 mM, pH 7.4), and diluted to ∼105 CFU per well. Frozen A. f umigatus spores were diluted to ∼105 CFU per well and grown in 1.9% malt extract. First, 50 μL of Tris buffer was added to each well and mixed with 50 μL of peptide solution (640 μM), and the mixtures in each well were serially diluted 2-fold. Then 50 μL of microorganism culture was added to 96-well plates, making 50 000 bacteria per well, and the plates were incubated at 37 and 27 °C, respectively, for 5 h. Then 5 μL of 20% TSB was added to each well, and the plates were incubated for 6−12 h depending on the organism. Experiments were performed in triplicate on two separate occasions, and the average MIC was calculated. Fluorometric Microculture Cytotoxicity Assay. The fluorometric microculture cytotoxicity assay (FMCA) is based on hydrolysis of fluorescein diacetate (FDA) by cells with intact plasma membranes. The assay procedures have been described previously.22

was dissolved in 50% CH3CN in H2O, containing 0.05% TFA, and freeze-dried. The crude peptide (102.2 mg) was purified using RPHPLC on a Phenomenex 250 × 21.2 mm C18 column packed with 10 μm particles at a flow rate of 10 mL/min, with a linear gradient (5− 100% solvent B; solvent A: 10% CH3CN + 0.05% TFA, solvent B: 60% CH3CN + 0.05% TFA) over 70 min. Oxidative Folding. The reduced pure peptide was dissolved in H2O (1 mg/mL) and transferred into folding mixtures containing different concentrations of 0.1 M NH4HCO3 (pH 8.5), i-PrOH, reduced glutathione (GSH), or oxidized glutathione (GSSG) (see Table 1 for details). After addition of folding reagents, samples were flushed with N2 and incubated for 24 h at room temperature. Condition #8 (0.1 M NH4HCO3 30% v/v and i-PrOH 60% v/v) was then used to test time intervals of 2 min, 1 h, 2 h, 4 h, 8 h, and 24 h and was quenched using double volumes of 5% HCOOH in 10% CH3CN (pH 1.7). Samples were analyzed using LC-MS. The rest of the peptide was folded in 0.1 M NH4HCO3 containing 60% (v/v) iPrOH for 2 min and purified by RP-HPLC on a Waters XBridge BEH300 10 × 250 mm 5 μm C18 column at a flow rate of 4 mL/min, using a linear gradient of 5% to 80% CH3CN in 0.05% TFA over 65 min. NMR Spectroscopy. Freeze-dried peptide (1.2 mg) was dissolved in 600 μL of H2O/D2O (9:1, v/v) at pH ∼5. 2D spectra were recorded at 290 and 298 K. All data, including TOCSY (mixing time 80 ms), NOESY (mixing time 150 ms), 13C-HSQC, 15N-HSQC, and DQF (double-quantum-filtered)-COSY were recorded and processed using Topspin (Bruker). The water signal was suppressed using a modified WATERGATE sequence.16 Generally, 4096 data points were collected in the F2 dimension and 256 (128 complex) points in F1, with 512 increments of 8 scans over 11 194 Hz. A series of TOCSY spectra were recorded at 290 K for hydrogen−deuterium exchanging experiments. Structure Calculations. Resonance assignments of RCB-1 were obtained using sequential assignment strategies. Once the chemical shift assignments were identified from TOCSY spectra, all NOE connections were identified and converted into interproton distances using CYANA 3.0.17 TOCSY, NOESY, DQF-COSY, and HSQC spectra were analyzed using the program package CARA.18 Peptide backbone φ/ψ torsion angle constraints were generated from TALOSN19 chemical shift analysis. Hydrogen bond restraints were identified by long-range NOE correlations and D2O exchange experiments in conjunction with preliminary structures. Solution structure calculations were performed using CYANA 3.0, and 50 random conformers were annealed in 8000 steps using torsion angle dynamics; 15 conformers with the lowest energies and the lowest residual restraint violations were selected to represent the solution structure of RCB-1. The 3D structures were generated using MOLMOL,20 and structure qualities were validated using MolProbity.7 The structures of RCB-1 and related chemical shifts have been deposited in the Protein Data Bank (RCSB PDB) and the Biomagnetic Resonance Bank (BMRB) as accession codes 2mtm and 25169, respectively. Serum Stability Assay. The serum stability assay was carried out in male human serum (Sigma-Aldrich, USA). The serum was centrifuged at 13 000 rpm for 10 min to remove lipids, and the supernatant was incubated at 37 °C for 10 min. Each assay was performed by individually adding 8 μL of 200 μM peptide to 80 μL of serum and then incubating at 37 °C for different time periods (0, 1, 6, and 24 h). The serum aliquot was quenched with 80 μL of 6 M urea and incubated at 4 °C for 10 min. Then the aliquot was mixed with 80 μL of 20% TCA and incubated at 4 °C for 10 min to precipitate serum proteins. The supernatant was collected after centrifugation at 13 000 rpm for 10 min, and 25 μL of the supernatant was analyzed by LC-MS, using a Shimadzu LC-10 HPLC system connected to a ThermoFinnigan (San Jose, CA, USA) LCQ Deca ESI ion trap MS operated in the positive mode. Separation was done on a Waters Xterra MS C8 2.1 × 150 mm 3.5 μm column at a flow rate of 0.3 mL/min, using a linear gradient of 5% to 90% CH3CN in H2O in 0.05% HCOOH for 30 min. The percentage of the remaining peptide in the serum was quantified by the integration of the area under the curve for the required peptide

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 184715031. Fax: +46 18509101. E-mail: ulf. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.B. was supported by the Maheva Programme (financed by Erasmus Mundus Action 2, 2010-2372/001-001-EMA2). U.G. was supported by grants from the Swedish Foundation for Strategic Research (F06-0058) and the Swedish Research Council (621-2007-567). H.R.E. is supported by a UM/ MOHE/HIR Grant (F000009-21001) from the Ministry of Higher Education, Malaysia. The authors would like to thank Dr. C. Persson for acquiring the NMR data, and the Swedish NMR Centre (Gothenburg) for access to NMR. We also would like to thank Drs. J. Levenfors and J. Bjerketorp for kindly providing us plant pathogens, Dr. R. Burman for performing the cytotoxicity assay, and Dr. A. Strömstedt for helping to transport plant pathogens and for assistance with antibacterial assays.

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REFERENCES

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