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ISOLATION AND CHARACTERISATION OF NOVEL CYCLOTIDES FROM

VIOLA HEDERACEAE: SOLUTION STRUCTURE AND ANTI-HIV ACTIVITY OF

VHL-1, A LEAF-SPECIFIC-EXPRESSED CYCLOTIDEBin Chen‡§, Michelle L. Colgrave‡, Norelle L. Daly‡, K. Johan Rosengren‡,

Kirk R. Gustafson¶ and David J. Craik‡From the ‡Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072,

Australia; the §Centre for Natural Products, Chengdu Institute of Biology, Chinese Academy of

Sciences, Chengdu 610041, P. R. China; the ¶Molecular Targets Development Program, Centre

for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA

Address correspondence to: David J. Craik, Institute for Molecular Bioscience, University ofQueensland, Brisbane, QLD 4072, Australia, Tel: 61-7-33462019; Fax: 61-7-33462029; Email:d.craik@imb.uq.edu.au

Based on a newly established

sequencing strategy featured by its efficiency,

simplicity and easy manipulation, the

sequences of four novel cyclotides

(macrocyclic knotted proteins) isolated from

an Australian plant Viola hederaceae were

determined. The three-dimensional solution

structure of vhl-1, a leaf-specific-expressed

31-residue cyclotide, has been determined

using two-dimensional1H-NMR spectroscopy.

Vhl-1 adopts a compact and well-defined

structure including a distorted triple-

stranded β-sheet, a short 310 helical segment

and several turns. It is stabilised by three

disulfide bonds, which, together with

backbone segment, form a cyclic cystine knot

motif. The three-disulfide bonds are almost

completely buried into the protein core, and

the six cysteines contribute only 3.8% to the

molecular surface. A pH titration experiment

revealed that the folding of vhl-1 shows little

pH dependence and allowed the pKa of 3.0 for

Glu3 and ~5.0 for Glu14 to be determined.

Met7 was found to be oxidised in the native

form, consistent with the fact that its side

chain protrudes into the solvent, occupying

7.5% of the molecular surface. Vhl-1 shows

anti-HIV activity with an EC50 value of 0.87

μM.

Cyclotides are a recently characterizedfamily of naturally occurring circular mini-proteins of 28 to 37 amino acid residues isolatedfrom plants of the Rubiaceae and Violaceaefamilies (1, 2). Their head-to-tail backbone andsix conserved cysteine residues make up atopologically unique structure designated as acyclic cystine knot in which two disulfide bondsand their connecting backbone segments form anembedded ring that is penetrated by a thirddisulfide bond (3). Fig. 1 shows the structure ofkalata B1, the first example characterised in thecyclotide family, and its sequence is presented in

Table 1. The fact that cyclotides areexceptionally resistant to thermal and proteolyticdegradation has led to the suggestion that thecyclic cystine knot motif contributes greatly totheir stability. By contrast, the amino acidsequences between the six cysteines, which formsix loops that present on the surface of themolecules, affect the surface characteristics andbiological activities of the cyclotides. Thecyclotides have been divided into twosubfamilies, Möbius and bracelet cyclotides (1,4) based on the presence or absence,respectively, of a cis-Pro peptide bond in thecircular peptide backbone.

The cyclotides show a diverse range ofbiological activities, including uterotonicactivity of kalata B1 from Oldenlandia affinis

DC (5, 6), the HIV inhibitory activity ofcirculins from Chassalia parvifolia Schum,cycloviolins from Leonia cymosa Mart., andpalicourein from Palicourea condensata Standl.(7-9), the antimicrobial activity of kalata B1,circulins A and B, and cyclopsychotride A fromPsychotria longipes Muell. Arg. (10), thecytotoxic activity of cycloviolacin O2 fromViola odorata L. and vitri A from Viola tricolor

L. (11, 12), the neurotensin antagonistic activityof cyclopsychotride A (13), the hemolyticactivity of violapeptide I from Viola tricolor L.(14), the trypsin inhibitory activity of MCoTI-Iand II from Momordica cochinchinensis (15),and the insecticidal activity of kalata B1 andkalata B2 (16, 17). All of these cyclotides wereoriginally found through screening programs forbiological activities, or, in the case of theuterotonic activity from native medicine usage(5). Some cyclotides, for example, kalata B1,circulins and cyclopsychotride A, effectivelyshow several different inhibitory activities,suggesting that the loops of one cyclotidemolecule may have various biological functionsand can potentially bind to different target sites.Perhaps most interesting is the finding that

JBC Papers in Press. Published on April 11, 2005 as Manuscript M501737200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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kalata B1 and kalata B2 inhibit the growth anddevelopment of Helicoverpa punctigera larvae,leading to the suggestion that cyclotides play animportant role in plant defence against pests orpathogens (16, 17). The diverse bioactivities,along with their unique structural characteristics,render the cyclotides extremely important interms of potential agrochemical orpharmaceutical applications (4, 16, 18).

The cyclotides, like many other defenceproteins, are derived from precursorpolypeptides, which undergo a process of post-translational modification. Recently we reportedthe isolation and characterization of cyclotidecDNA clones from Viola odorata (19). A singlefull-length cDNA transcript encodes one orseveral cyclotides, which are separated byconserved peptide fragments termed N-terminalrepeats (NTR) that may function in thecyclization and folding of cyclotides (19). Wealso synthesized the NTRs based on the cDNAclones and elucidated their α-helical solutionstructures by NMR spectroscopy. Interestinglythe NTR sequences were not as conserved asthose predicted from cDNA clones fromOldenlandia affinis (16). This might result fromthe fact that the cDNAs from V. odorata encodeboth subfamilies, Möbius and braceletcyclotides, instead of representing mainly onesubfamily of cyclotides, as was the case for thecDNAs isolated from O. affinis (16, 19).

To date, approximately 50 cyclotideshave been reported from a number of species ofthe Rubiaceae and Violaceae plant families, andall have molecular weights ranging from 2.8 to3.5 kDa. A recent study by HPLC and LC-MSon cyclotides from Viola hederaceae revealedthat up to 66 different masses, most likelycorresponding to the same number of cyclotides,were discerned in extracts of various parts of theplant (20). This suggests that the cyclotides are avery diverse family of proteins and potentiallyare much more common in plants than hadpreviously been realised. To expand ourknowledge of structure-activity relationships ofthe cyclotides, we introduce a new strategy fordetermining the sequence of cyclic disulfide richpeptides, report the isolation and characterisationof four novel cyclotides from V. hederaceae andpresent the solution structure of vhl-1, one of thenew cyclotides.

EXPERIMENTAL PROCEDURES

Isolation and Purification of vhl-1 - Fresh wholeplant of V. hederaceae was collected from agarden in Brisbane, Australia, and separated into

different parts: leaves, petioles, flowers,pedicels, roots, bulbs, as well as above andbelow ground runners. The leaves werehomogenized using a blender (Moulinex) andextracted with dichloromethane:methanol (1:1)overnight. Plant debris was removed using acotton plug. The filtrate was repeatedlypartitioned with dichloromethane and water. Theorganic soluble fraction was discarded, and themethanol/water layer was concentrated on arotary evaporator (Bücchi) prior tolyophilization. Then the mixture was dilutedwith distilled water to a final methanolconcentration < 20% and lyophilized on afreeze-drier (Speedvac). The dried material wasredissolved in a minimal amount of buffer A(0.05% trifluoroacetic acid prepared in distilledwater). The solution was then passed through asolid phase filter (Sartorius) before purificationusing preparative RP-HPLC on an Agilent 1100series system with variable wavelength detectorand Phenomenex Jupiter C18 column (250 × 22mm, 5 μm, 300Å). Gradients of buffer A (0.05%aqueous trifluoroacetic acid) and buffer B (90%acetonitrile, 0.05% trifluoroacetic acid) wereemployed with a flow rate of 8 ml/min and agradient of 1% buffer B per minute. Furtherpurification was performed using semi-preparative RP-HPLC on a Phenomenex JupiterC18 column (250 × 10 mm, 5 μm, 300Å) andusing an analytical Phenomenex Jupiter C18column (250 × 4.6 mm, 5 μm, 300Å). The finalpurity was examined with analytical RP-HPLCon a Grom column (150 × 2 mm, 3 μ m ,equipped with a Security-guard column) with aflow rate of 300 μl/min. Masses were analysedon a Micromass LCT mass spectrometerequipped with an electrospray ionization source.Aminoethylation of cysteines - The reduction andalkylation of the disulfide bonds was performedaccording to the method described in theliterature (21) with minor modification. 5 nmolof vhl-1 was reduced with 0.4 μmol DTT in 200μ l 0.25 M Tris-HCl, containing 1 mM EDTAand 8 M guanidine-HCl (pH 8.5, 37 ºC) underN2. After 2 hours, 20 μmol bromoethylaminedissolved in 20 μl 0.25 M Tris-HCl, containing 1mM EDTA and 8M guanidine-HCl (pH 8.5) wasadded. The reaction was incubated in a waterbath at 37 ºC in the dark under N2 overnight andterminated by injection onto RP-HPLC andeluted with a linear gradient of 0-80% buffer Bin 80 minutes. The molecular weights of thecollected fractions were confirmed by LCT-ESI-MS prior to lyophilization and storage at -20 ºC.

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Reduction of vhl-1 and MALDI-MS analysis - To~6 nmol of vhl-1 in 20 μl of 0.1M NH4HCO3

(pH 8.0), 1 μl of 0.1M TCEP was added and thesolution was incubated at 65 ºC for 10 min. Thereduction was confirmed by MALDI-TOF-MSafter desalting using Ziptips (Millipore), whichinvolved several washing steps followed byelution in 10 μ l of 80 % acetonitrile (0.5 %formic acid). The desalted samples were mixedin a 1:1 ratio with matrix consisting of asaturated solution of α - c y a n o - 4 -hydroxycinnamic acid in 50% acetonitrile (0.5%formic acid). The instrument used was aVoyager DE-STR mass spectrometer (AppliedBiosystems). 200 shots per spectra wereacquired in positive ion reflector mode. Thelaser intensity was set to 1800, the acceleratingvoltage was set to 20,000 V; the grid voltage setto 64% of the accelerating voltage and the delaytime was 165 ns. The low mass gate was set to500 Da. Data were collected between 500 and5000 Da. Calibration was undertaken using apeptide mixture obtained from Sigma Aldrich(MSCal1).Enzymatic Digestion and nanospray MS-MS

sequencing - To the reduced peptide, trypsin,endoGlu-C or a combination of both were addedto give a final peptide-to-enzyme ratio of 50:1.The trypsin incubation was allowed to proceedfor 1 hr, the endoGlu-C was over 3 hr, whilst forthe combined digestion trypsin was addedinitially for 1 hr followed by the addition ofendoGlu-C for a further 3 hr. The digestionswere quenched by the addition of an equalvolume of 0.5% formic acid and desalted usingZiptips (Millipore). Samples were stored at 4 ºCprior to analysis. The fragments resulting fromthe digestion were examined first by MALDI-TOF-MS followed by sequencing by nanosprayMS-MS on a QStar mass spectrometer. Acapillary voltage of 900 V was applied andspectra were acquired between m/z 60-2000 forboth TOF spectra and product ion spectra. Thecollision energy for peptide fragmentation wasvaried between 10-50 V, depending on the sizeand charge of the ion. The Analyst softwareprogram was used for data acquisition andprocessing. The MS-MS spectra were examinedand sequenced based on the presence of both b-and y-series of ions present (N- and C-terminalfragments). The same procedure was utilized forthe sequencing of the three other novelcyclotides isolated from V. hederaceae.Chymotrypsin digests using the same conditionsas for trypsin were also conducted to confirm the

results obtained for each of the peptidesequences.NMR experiments – The sample for NMRspectroscopy was prepared by dissolving vhl-1in 70% H2O, 25% CD3CN and 5% D2O to a finalconcentration of 1.4 mM, since aggregation ofthe peptide was observed in 100% water. Allspectra were recorded on Bruker ARX 500 orBruker ARX 600 spectrometers equipped with ashielded gradient unit, with sample temperaturein the range 283-330 K. All spectra wereacquired in phase-sensitive mode using timeproportional phase incrementation (22). ForTOCSY (23), using MLEV-17 (24) with amixing time of 80 ms, and NOESY (25) withmixing times of 100, 200 and 250 ms, watersuppression was achieved using aWATERGATE (water suppression by gradient-tailored excitation) (26) sequence. Doublequantum-filtered COSY (27) and E-COSY (28)were also recorded. Slowly exchanging amideprotons were identified by recording a series ofone-dimensional spectra and two-dimensionalTOCSY spectra at 298 K over a period of 20 himmediately after dissolution of a sample in25% CD3CN and 75% D2O.

The pH dependence was monitored forvhl-1 at 298 K by altering the sample pH from2.0 to 6.0 by adding HCl or NaOH. The pKa ofthe titrating Glu3 and Glu14 residues wasdetermined for vhl-1 by nonlinear curve fittingof the data points. The 3

JHN-Hα coupling constantswere obtained from a high-resolution one-dimensional spectrum and from line-shapeanalysis of the anti-phase cross-signal splittingin a high-resolution DQF-COSY spectrum.

All two-dimensional spectra werecollected over 4,096 data points in the f2dimension and 512 increments in the f1 andprocessed using XWINNMR (Bruker) on aSilicon Graphics Octane workstation. The f1dimension was generally zero-filled to 2,048 realdata points, with the f1 and f2 dimensions beingmultiplied by a sine-squared function prior toFourier transformation. Chemical shifts wereinternally referenced to sodium 2,2-dimethyl-2silapentane-5-sulfonate.Structural restraints - Distance restraints werederived primarily from cross-peaks in a 250 msmixing time NOESY spectrum recorded at 298K. The cross-peaks were analysed and resonanceassignments were achieved using the programSPARKY (29). 25 backbone dihedral restraintswere added on the basis of 3

JHN-Hα couplingconstants derived from the splitting of the amideand α protons, and were constrained to –120

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(±30)° for 3JHN-Hα in the range 8.5±0.5 Hz

(residues 1, 3, 4, 5, 7, 8, 10, 19, 21, 24, 30), -120(±15)° for 3

JHN-Hα greater than 9.5 Hz (residues26, 27) and –65 (±30)° for 3

JHN-Hα less than 5.8Hz (residues 6, 9, 11, 14, 15, 16, 31). Oneadditional constraint of –100 (±80)° (residue 18)was applied where the 3

JHN-Hα coupling constantis ~7.00 Hz and the intraresidue Hαi-HNi NOEis weaker than the sequential Hα i-HNi NOE.Residues 22, 23, 28 and 29 with intense Hαi-HNi

NOEs and 3JHN-Hα coupling constants of ~7 Hz

were restrained to 50 (±40)°. 3JHα-Hβ coupling

constants derived from an ECOSY spectrum,together with NOE intensity patterns from theNOESY spectrum with a mixing time of 100 ms,were used to determine the stereospecificassignments of β–methylene protons and 17 χ1dihedral angles restraints. Ten hydrogen bondswere determined based on slow exchange dataand preliminary structure calculations. 20restraints of 1.7-2.2 and 2.7-3.2 Å for thesehydrogen bonds were added and used in the finalstructure calculation.Structure calculation - Initial structures werecalculated using DYANA (30) based on NOEdata output from SPARKY. After an iterativeprocess in which preliminary structures wereused to resolve ambiguities, sets of 50 structureswere calculated using a torsion angle simulatedannealing protocol within the program CNS(31). This protocol involved a high temperaturephase comprising 4,000 steps of 0.015 ps oftorsion angle dynamics, a cooling phase with4,000 steps of 0.015 ps of torsion angledynamics during which the temperature waslowered to 0 K, and finally an energyminimization phase comprising 500 steps ofPowell minimization. The resultant structureswere subjected to further molecular dynamicsand energy minimization in a water shell (32). Aset of 20 structures with the lowest overallenergy that had no violations of distancerestraints greater than 0.2 Å or dihedral anglerestraints greater than 3º was chosen to representthe structure of vhl-1. Structures were visualisedusing the program MOLMOL (33) and analysedwi th PROMOTIF_NMR ( 3 4 ) a n dPROCHECK_NMR (35). The 20 final structuresof vhl-1 and associated restraints have beendeposited in the protein Data Bank (ID code:1ZA8).

RESULTS

Isolation and purification of cyclotides

Cyclotides show several defining characteristics,including late elution on HPLC and a mass rangefrom 2.8 kDa to 3.5 kDa. Based on thesecharacteristics, we have established an effectiveand efficient procedure to isolate and purifycyclotides from various components of the crudeplant extracts (16). In the current study, wedivided fresh collected plant material into eightparts (leaves, petioles, flowers, pedicels, aboveground runners, below ground runners, bulbsand roots) and used the extraction protocol toobtain crude cyclotide extracts. RP-HPLCprofiles of different parts of V. hederaceae areshown in Fig. 2. Repeated RP-HPLC resulted inthe separation and purification of four novelcyclotides corresponding to chromatographicpeaks 4, 5 (two peptides) and 6. The molecularweights of these cyclotides determined as shownin Table 1 by electrospray ionization (ESI) massspectrometry revealed they were new whencompared to the molecular weights of knowncyclotides. The cyclotide corresponding tochromatographic peak 3 is expressed only inleaves, but its mass of 3117.8 matches a reportedcyclotide cycloviolacin O10 isolated from Viola

o d o r a t a (1). Chromatographic peak 7corresponds to a cyclotide named vhr1, whosemolecular characterisation was reported recently(20). Among the four novel cyclotides, the twocorresponding to peak 5 are expressed in severaldifferent tissues and were designated ascycloviolacin H2 and H3. The one with arelatively late retention time corresponding topeak 6 is only expressed in leaves and named asvhl-2 (Viola hederaceae leaf cyclotide-2).

Special emphasis was put on a cyclotidevhl-1 (Viola hederaceae leave cyclotide-1) witha retention time of 32.0 minutes (peak 4) as it isrelatively abundant and expressed specifically inleaves and not in any other parts of the plant.From the crude extract of V. hederaceae leavesvhl-1 was isolated and purified to homogeneityby RP-HPLC as shown in the last panel in Fig.2. The yield of vhl-1 was about 1 mg/kg freshplant material.Amino Acid Sequence Analysis

Amino acid analysis of vhl-1 indicated that it iscomposed of 31 amino acids: 1 Ala, 6 Cys, 2Glu or Gln, 2 Phe, 2 Gly, 3 Ile, 2 Lys, 1 Leu, 1Met, 2 Asp or Asn, 5 Ser, 1 Thr, 2 Val and 1Tyr. It shows resistance to enzymatic cleavageby trypsin, consistent with the presence of acyclic cystine knot motif characteristic of allcyclotides. After reduction and alkylation, eachreduced and S-aminoethylated cysteinecontributes to an increment of molecular weightby 44 Da. Nanospray-MS and LC-ESI-MS

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analysis of the native (3330, mono-isotopicmass) and S-aminoethylated peptide (3594)demonstrated that, like other cyclotides, sixcysteines are present in three intramoleculardisulfide bonds. Tryptic cleavage was thencarried out on the alkylated peptide in an attemptto use a recently proposed ‘loop sequencing’method for determining the primary structure ofcyclotides (21, 36). However, this approachresulted in very complicated enzymaticfragments, implying that, besides the sixalkylated cysteines, several other positivelycharged amino acid residues exist in the primarystructure. All of the alkylated cysteines andpositive residues provide cleavage sites fortrypsin and, as a result, produce additionalfragments, complicating the MS-MS data.

An alternative strategy outlined in Fig.3, which includes both enzymatic digestion andMS-MS fragmentation, was used to complete thesequence analysis. The native cyclotide wasreduced by TCEP and subjected to enzymaticdigestion directly, without alkylation of thecysteines. The reduction of the peptide wasconfirmed by the observation of peaks at m/z

11123+ and 8344+, which correspond to amolecular mass of 3336 Da due to the additionof six protons to the native peptide. Digestion ofthe reduced peptide with endoproteinase GluC(EndoGluC) gave rise to two major peaks at m/z

627.72+ and 707.33+ corresponding to twofragments with masses of 1253.5 and 2118.9 andimplying the presence of two glutamic acidresidues. Several other peaks with relatively lowintensities were also produced after digestion bytrypsin or a combination of trypsin andEndoGluC.

The study of the MS-MS fragmentationof the first fragment at m/z 627.72+ resulted incompletion of its amino acid sequence as shownin Fig. 4A , suggesting that it consists of 11amino acid residues with the sequenceSCAF*ISFCFTE. The b2 ion of the fragment atm/z 191 could be either SC or CS. However, itwas determined to be SC by comparison withknown cyclotides and further confirmed byNMR spectroscopy. The second fragment at m/z

707.33+ was difficult to analyse by MS-MSfragmentation owing to its length. However, twopartial sequences from both N-terminal(VIGCSCKNKV-) and C-terminal (-LNSISCGE) were observed as highlighted inFig. 4B and in the supplementary Table 1. Theb- and y-ions greater than ~5% in relativeintensity are labelled in Fig. 4 and a number ofions that allow full sequence elucidation are alsopresent in low abundance (see supplementary

Table 1). The third enzymatic fragment resultingfrom digestion by trypsin and EndoGluC at m/z

715.32+ was selected and subject to MS-MSfragmentation. Fig. 4C indicates that thesequence NKVCYLNSISCGE was deduced,which is actually a part of the second fragmentand, together with the information of the N-terminal sequence of the second fragment, led tothe establishment of the sequenceVIGCSCKNKVCYLNSISCGE (m/z 707.33+) ofthe second fragment.

The amino acid composition of theproposed sequence formed by combining thethree fragments is in agreement with the aminoacid analysis, except for the residue Met. Themass difference between ion b4 and b3 infragment 1 is 147 Da, suggesting that the aminoacid residue is Phe (F) as marked *. However, intwo dimensional TOCSY and NOESY spectra ofvhl-1, the spin system of this residue shows thepresence of two β and two γ protons, none ofwhich have any correlations to aromatic protonsas shown in Fig. 5A. This result, along with thepresence of Met from amino acid analysis andthe information that this residue has a mass of147 Da (16 Da addition of 131 Da for Met),indicates that this residue is a mono-oxidisedMet, instead of Phe. This conclusion is fullyconsistent with the results of amino acid analysisand the ‘sequential walk’ in NOESY spectra.The identification of the two fragments derivedf r o m t h e E n d o G l u C d i g e s t i o n ,VIGCSCKNKVCYLNSISCGE (m/z 707.33+)and SCAM*ISFCFTE (m/z 627.72+) rather thanSCAF*ISFCFTE was therefore successfullycompleted.

To determine whether the mono-oxidised Met exists in the native cyclotide or isoxidised during the process of isolation andpurification, we extracted fresh leaves usingmethanol under N2 for different time periods andinvestigated the mass of the cyclotide byMALDI-MS spectrometry. The mass of 3330was detected in samples extracted quickly undernon-oxidising conditions, suggesting that themethionine exists naturally in oxidised form inthe plant.

The sequences of the other three novelcyclotides vhl-2, cycloviolacin H2 and H3 asshown in Table 1 were also established using asimilar procedure.Structure elucidation of Vhl-1

The NMR solution structure of vhl-1 wasdetermined based on homonuclear 2D NMRexperiments including TOCSY, NOESY, DQF-COSY and E-COSY. As shown in Fig. 5, thecross peaks in the 2D NMR fingerprint regions

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are well dispersed and could be unambiguouslyidentified for spin system assignments in theTOCSY spectrum and for sequentialconnectivities in the NOESY spectrum. Theunbroken sequent ia l -walk conf i rmsunequivocally the head-to-tail circular backboneof the peptide and the amino acid sequence fromMS-MS fragmentation and amino acid analysis.The structure was calculated from a total of 232interproton distances derived from NOESYspectra, including 103 sequential, 51 medium-range and 78 long-range NOE restraints. On thebasis of coupling constants, 25 φ and 17 χ1dihedral angle restraints were included in thestructure calculations. Finally, 20 upper-limithydrogen bond restraints for 10 hydrogen bondsderived from amide exchange experiments andpreliminary structures were included.

A set of 50 structures was calculatedusing torsion angle simulated annealing. The 20structures with the lowest overall energy thathad no violations of distance restraints greaterthan 0.2 Å or dihedral angle restraints greaterthan 3˚ were chosen to represent the structure ofvhl-1. Fig. 6A shows the NMR ensemble of thefinal 20 energy-minimized conformers and thestructural statistics for this family aresummarised in Table 2. The mean globalbackbone rmsd is 0.28 Å for residues involvedin secondary structure elements and 0.32 Å forall residues. For heavy atoms the rmsd is 1.14 Åfor all residues. The small rmsd values over themolecules for the backbone and heavy atoms ofall the residues, as well as the minimal restraintviolations, shows that vhl-1 adopts a well-defined structure.

The peptide backbone of vhl-1 is foldedback onto itself and is stabilised by threedisulfide bonds. The three-dimensional structurecomprises a central β sheet region (colouredcyan in Fig. 6B), consisting of residues Cys18-Lys21 in loop 4 and Val24-Leu27 in loops 5/6,which, together with a type I′ β-turn centering atresidues Asn22 and Lys23, forms a β-hairpin.This motif, combined with a third distortedstrand involving residues Glu3, Ser4 and Cys5,makes up a triple-strand antiparallel β-sheetsflanked by a short 310 helix (Glu14-Ile16) inloop 3. Two additional type I′-like β-turns arepresent in the regions of Ala6-Ser9 and Leu27-Ile30. The extended loop 2 protrudes onto thesurface of the molecule, while loop 6 is twisted~90° relative to the triple-strand antiparallel β-sheet base. The secondary structural elementsare recognized in a majority of the 20 lowest

energy structures by the program PROMOTIF(34).Disulfide connectivity pattern

Previous studies suggested a common pattern forthe disulfide bond connectivity of I-IV, II-V, III-VI in cyclotides (6). This connectivity wasfurther confirmed based on the extensiveinvestigation of χ1 dihedral angles of thecysteine residues and direct chemical evidenceusing a novel approach for disulfide analysis,involving partial reduction and stepwisealkylation (36, 37). In this study, we determinedthe χ1 dihedral angle restraints for the sixcysteines by analysing the 3

JHα-Hβ couplingconstants and NOEs intensities, all of which areconsistent with those of kalata B1, cycloviolacinO1 and vhr1 (20, 37). Therefore, we used thispattern of disulfide connectivity as shown in Fig.6B , a knotted arrangement, in the structurecalculations, instead of the laddered onesuggested as an alternative (38). The peptidestructure obtained is in excellent agreement withthese experimental restraints.Determining the pKa values of Glu-3 and Glu-14

of vhl-1

The cyclotide vhl-1 contains two glutamic acidresidues, namely Glu3 and Glu14. The formerresidue is absolutely conserved throughout thecyclotide family, suggesting a crucial structuralor functional role. Their pK a values weredetermined by monitoring the chemical shifts ofother nearby protons (39). Recording TOCSYspectra as a function of pH revealed that themajority of amide chemical shifts fluctuate byless than 0.1 ppm over the pH range 2-6,indicating that the protonation or deprotonationof carboxylate groups does not affect the overallstructure. However, the amide protons ofresidues Phe12 and Thr13 shifted downfield bymore than 3 and 1.5 ppm, respectively, over theindicated pH range as shown in Fig. 7A. Similarshifts were also observed in kalata B1 andcycloviolacin O1 (37). This is because as pHincreases, the deprotonated carboxylate of Glu3functions as a proton acceptor to form hydrogenbonds with the proximal amides of Phe12 andThr13, causing them to shift downfield. Fig. 7B

shows the hydrogen bond interaction betweenamides of Phe12 and Thr13 and carboxylate ofGlu3. In addition, the Thr13 hydroxyl protonwas observed in the NOESY spectrum recordedat pH 3.2, indicating that it is involved in a sidechain-side chain hydrogen bond interaction.These interactions account for the relatively lowpKa value of 3.0 for Glu3 (40). We also obtainedinformation on the pKa of Glu14 by analysing

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the titration curve for Cys11 shown in Fig. 7A.The Cys11 amide is slightly affected by thedeprotonation of Glu3 upon increasing pH from2.0 to 4.0 and shifts upfield by 0.5 ppm. Fig. 7C

shows the relative orientation of this amide. Oninteraction with the deprotonated carboxylategroup of Glu14 on increasing pH from 4.0 to6.0, the amide of Cys11 shifts back downfield by~0.4 ppm, displaying a second sigmoidal curveand allowing a pKa value of ~5.0 for Glu14 to bedetermined. Compared with the pKa value of 4.5as expected for a Glu carboxylate in a randompeptide, this slight pKa shift may be caused byelectrostatic interactions of this residue with theproximal Glu3 (41).

DISCUSSION

Although cyclotides were firstdiscovered in a limited number of Rubiaceaeplant species, recent studies have revealed theyare widespread in the plant kingdom andespecially abundant in the genus Viola of thefamily Violaceae (1, 12, 14, 21, 42). Moregenerally, circular proteins of various types havebeen discovered in bacteria, plants and animalsin recent years (2). Some cyclotides can beexpressed in different plants, or even in differenttissues of one plant species, as is the case forHPLC peaks 1 and 2 of V. hederaceae shown inFig. 2, which correspond to cyclotides kalata B1and kalata S, respectively. In fact, these twocyclotides constitute two main components inalmost all parts of the plant except in flowers.However, other cyclotides are expressed only inone or several tissues, for example, in aerialparts. In this study, attention was focused onthose cyclotides that occur in only one planttissue, because these molecules might haveunique structures, bioactivities or physiologicalfunctions.

As can be seen in Fig. 2, the RP-HPLCprofile of leaves of V. hederaceae is quitedifferent from those of other tissues in that thereare three tissue-specific cyclotides (peaks 3, 4and 6). Repeated RP-HPLC on the crude extractsof V. hederaceae leaves resulted in the isolationof vhl-1 as well as three other novel cyclotidesvhl-2, cycloviolacin H2 and cycloviolacin H3.Amino acid analysis and sequencing clearlyindicated that vhl-1 is composed of 31 aminoacid residues and shares the same highlyconserved six cysteine residues as all othermembers of the cyclotide family, suggesting thatit adopts a similar three-dimensional structure. Itbelongs to the bracelet subfamily of cyclotidesdue to the absence of a cis-Pro peptide bond in

loop 5 (1). Comparison of the vhl-1 sequencewith known cyclotides allows some conclusionsto be drawn regarding the six successive loops.The peptide possesses highly conserved loops 1and 4, which consist of three residues, GES, andone residue, S, respectively. This conservationlikely occurs because loops 1 and 4 form thebackbone segments of the embedded ring of thecyclic cystine knot and their amino acid residuesplay significant roles in the structural stabilityand bioactivities of cyclotides (data not shown).Loop 5 contains four residues, KNKV, with twopositively charged residues in close proximity,which is in agreement with 11 known cyclotidesin the bracelet subfamily.

However , vhl-1 shows somedistinguishing characteristics in terms of itsamino acid composition and sequence. Itpossesses an extended loop 2, with five aminoacid residues AM*ISF, compared with fourresidues in loop 2 of all other cyclotidescharacterised so far. Vhl-1 is also the firstcyclotide containing an oxidised Met. Massspectral investigation of vhl-1 by MALDI-MSconfirmed the cyclotide contains the mono-oxidised derivative of Met with a mass of 3330,instead of 3314 Da. Loop 3, comprising sixresidues FTEVIG, shows the common feature ofa majority of residues being hydrophobic as wellas a relatively conserved hydroxyl-containingThr and highly conserved last residue Gly.However, it differs from other cyclotides in thatit contains a second Glu residue, whichcontributes an additional cleavage site forEndoGluC. Furthermore, loop 6 has an unusualcharacteristic that the last residue Ser replacesthe Pro shared by all other bracelet subfamilycyclotides. The structural roles of these residueswill be discussed later in relation to the structureof vhl-1.New strategy for sequencing analysis

Cyclotides are a unique, disulfide-rich family ofproteins with a circular backbone and cycliccystine knot motif, and as a consequence areimpervious to proteolytic digestion and can notbe directly sequenced by Edman degradation.Previous methods for amino acid sequencingwere all based on reduction and alkylation ofpeptides followed by enzymatic cleavage forEdman degradation or MS-MS analysis.Multiple handling and purification procedures inthese methods, as well as a tendency to fail toyield informative data made it imperative todevise a new strategy. In the current study, thesample preparation process for MS-MSfragmentation is very simple and practical. Theion suppression effect of TCEP in the reaction

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solution was easily diminished through the useof Ziptips (Millipore) for desalting. Theprocedure involving the reduction andsubsequent cleavage by one or more proteolyticenzymes occurs in one reaction vessel on ananomolar scale and requires only a singledesalting step followed by direct analysis byMALDI-MS and nanospray-MS or acombination of these two complementarytechniques. The resulting data were readilyinterpretable and allowed the rapid elucidationof the primary sequence of vhl-1, as well asother novel cyclotides, which were otherwisedifficult to sequence by existing methods.Sequence and structure homology with other

cyclotides

Sequence alignment of the cyclotides in Table 1indicates that in addition to six cysteines, someresidues such as Glu3 in loop 1 and Gly17 inloop 3 are extremely conserved in both Möbiusand bracelet subfamilies. Glu3 and Gly17 appearto be important because the Glu3 side chain,through extensive hydrogen bonding, forms apart of the folding scaffold (37). Gly17 isinvolved in a connecting site between helix andstrand for bracelet cyclotides and between turnand β -sheet for Möbius cyclotides. Anotherconserved residue throughout the cyclotidefamily is Asn (Asn28 for vhl-1) in loop 6, whichis likely involved in cleavage and cyclisationfrom the precursor protein to produce the maturepeptide. The additional Glu14 in loop 3 of vhl-1appears not to significantly affect the molecularfolding because its carboxylate group is solventexposed and has little capacity to form hydrogenbonds with internal amides. By contrast withother cyclotides, a hydrophilic loop 6 is present,since the NSIS sequence segment replaceshydrophobic residues shared by almost all othercyclotides. This segment twists and protrudesfrom the surface.

To date, although about 50 cyclotideshave been characterised, three-dimensionalstructures of only eight cyclotides namely kalataB1 and B2 (6, 17), circulins A and B (43, 44),cycloviolacin O1 (1, 37), palicourein (45), vhr1(20) and vhl-1 have been determined usingNMR techniques. The last six cyclotides belongto the bracelet subfamily and have six or sevenresidues in loop 3. These residues are involvedin a short 310 helical conformation, as is the casefor cycloviolacin O1, vhr1, palicourein and vhl-1. In contrast, kalata B1 and B2, the soleexamples from the Möbius subfamily that havebeen structurally characterised, contain only four

residues in loop 3, which are involved in a typeII β-turn (17, 37).

Like other known cyclotides, the overallstructure of vhl-1 adopts a compact fold withthree disulfide bonds involving formation of acystine knot buried into the protein core.Analysis of the surface-exposed residues showsthat the six cysteine residues contribute only3.8% to the surface of the molecule. However,hydrophobic residues constitute 57.3% of themolecular surface, compared to 39.0% for kalataB1 and 57.0% for vhr1 and cycloviolacin O1.These data explain the late HPLC retentiontimes of cyclotides compared to the majority ofother peptides. Despite the larger hydrophobicpatch on the molecular surface of vhl-1compared to vhr1, it eluted 2.5 minutes earlierthan vhr1 under the same chromatographicconditions. This is most likely because vhl-1contains an additional Lys and Glu, both ofwhich are exposed on the molecular surface.Among the 57.3% molecular surface constitutedby 19 residues including two Gly, 7.5% isattributed to Met7. This indicates that the sidechain of Met7 protrudes onto the molecularsurface, making it easily oxidised.

The surface representation of the vhl-1structure (Supplementary Information) revealsthat hydrophobic residues involving loops 2 and3 almost form a continuous surface, which isonly disturbed by a core close to Glu3. Lys21,Asn22 and Lys23 in loop 5 and Asn28, Ser29and Ser31 in loop 6, however, form ahydrophilic region punctuated by hydrophobicpatches from Val24 and Ile30.Implications for biological activities

Among the large number of cyclotidesdiscovered to date, only two cyclotides containMet. Varv B isolated from Viola arvensis (46)contains a Met in loop 5 as shown in Table 1.The reduced Met in Varv B suggests that theside chain may be involved in the hydrophobiccore and not solvent exposed. By contrast, theoxidised Met7 in vhl-1 results from its side chainprotrusion into the molecular surface, and mayplay a role in the physiological functions. Ingeneral, oxidation of Met residues in proteins isassociated with aging as well as in pathologicalconditions and results in increasing theirsusceptibility to proteolysis (47). But, it seemsthe latter is not the case for vhl-1 as there was noevidence for degraded forms in the extracts. Theoxidation of Met in proteins to Met sulfoxide isa reversible reaction mediated by methioninesulfoxide reductase (MsrA; EC 1.8.4.6) (48).One study suggested that oxidation andreduction of methionine could play a dynamic

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role in cellular signal transduction processes in avariety of systems (48), while other studies ledto another suggestion that Met residues mayserve as antioxidants and that MsrA acts toreduce the oxidised Met. MsrA and itscorresponding substrate---oxidised Met form anefficient means to repair oxidative damage in

v ivo (49, 50). Plants respond to virulentpathogens by producing active oxygen species,such as oxidised Met (51). Some pathogens, onthe other hand, can express MsrA to repairoxidative damage and as a result, can survive thehost environment (52). Whether vhl-1 plays sucha role amongst the suite of cyclotides found in V.

hederaceae remains to be determined.Vhl-1 was tested for anti-microbial

activity against several microorganisms(Escherichia coli, Staphylococcus aureus andCandida albicans), but showed no inhibitoryactivity against these microorganisms. Thisindicates that, if the theory that active-oxygenconstituents play a key role in plant defence istrue, the mechanism of peptides containingoxidised Met against pathogen invasion isdifferent from that applied to the anti-microbialbioassay. Vhl-1 was also screened for anti-HIV

activity and it inhibited the cytopathic effects ofHIV infection with an EC50 value of 0.87 μM. Arange of other cyclotides have been reported toexhibit anti-HIV activity with similar order ofmagnitude (53, 54). Clearly there is nophysiological reason for a plant to needprotection against HIV, but the anti-viral activitymay be an indicator of an as yet undiscoveredrole of the cyclotides as protective agents againstplant viruses.

Finally, the extremely stable andcompact structures of cyclotides, as well as theflexibility of both amino acid composition andsequences in loops 2, 5 and 6, provides a highlysuitable framework for drug design. Byanalysing structural features of a range ofdifferent cyclotides, it may prove possible tograft bioactive segments from other proteins orpeptides, which are normally unstable and havepoor bioavailability, into this framework, toform novel pharmaceutical molecules. In thecurrent study, we have enriched the knowledgeof the cyclotide family by characterising novelcyclotides and determining the solution structureof cyclotide vhl-1.

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85-94.46. Göransson, U., Luijendijk, T., Johansson, S., Bohlin, L.Claeson, P. (1999). J. Nat. Prod. 62: 283-

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(2001). Proc. Natl. Acad. Sci. USA 98:12920-12925.51. Hassouni, M. E., Chambost, J. P., Expert, D., Gijsegem, F. V., and Barras, F. (1999). Proc. Natl.

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FOOTNOTES

This work was supported by a grant from the Australian Research Council (ARC). D.C. is an ARCProfessional Fellow. B.C acknowledges the Chinese Academy of Sciences for visiting scholarship.The authors thank Horst Joachim Schirra for help with NMR experiments, Jason Mulvenna forhelpful comments in structure calculations, Jennifer Wilson for anti-HIV testing and Angela A. Salimfor anti-microbial testing.

The abbreviations used are: CCK, cyclic cystine knot; HPLC, high performance liquidchromatography; RP, reverse phase; LC-MS, liquid chromatography - mass spectrometry; MALDI-MS, matrix assisted laser desorption ionization mass spectrometry; NMR, nuclear magneticresonance; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlationspectroscopy.

FIGURE LEGENDS

Fig. 1. Ribbon representation of kalata B1 structure. The cyclotide framework consists of sixloops, separated by successive six cysteines, which are numbered in Roman numerals according totheir appearance order in the linear precursor protein. The cystine knot is made up by disulfide bondIII-VI penetrating the ring formed by disulfides I-IV and II-V and their connecting backbonesegments.

Fig. 2. Purification of vhl-1. RP-HPLC profiles of cyclotide extracts from various tissues (leaves;flowers; petioles; pedicels; above ground runners; below ground runners; bulbs and roots) of V.

hederaceae. 5 mg/ml solutions of crude cyclotide extracts from different plant parts were made andcentrifuged at 10, 000 g for 10 minutes. Then 8 μl supernatant samples were injected and analysed onan analytical C-18 Grom column (150 × 2 mm, 3 μm; flow rate: 300 μl per minute) with a linearacetonitrile gradient (10 to 80%) of buffer B (90% acetonitrile, 0.05% trifluoroacetic acid) in 40 min.The purity of vhl-1 shown in the last panel, obtained from the leaves of the plant, was assessed by RP-HPLC on the same analytical C-18 column using the same method.

Fig. 3. Strategy employed for amino acid sequence analysis. Disulfide bonds were reduced byTCEP. M*: mono-oxidised methionine.

Fig. 4. MS-MS fragmentation recorded by nanospray MS-MS spectrometry. The masses werecalculated on the basis of the mass-to-charge ratio. A, a double charged peak at m/z 627.72+

corresponding to the molecular weight 1253.5 Da of the first fragment. B, a triple charged peak at m/z

707.33+ matching the second fragment with a molecular weight of 2118.9 Da. C, a double chargedpeak at m/z 715.32+ representing the third fragment with a molecular weight of 1428.6 Da.

Fig. 5. TOCSY and NOESY spectra of vhl-1. Fingerprint regions of A, 80 ms TOCSY spectrumand B, 250 ms NOESY spectrum in 75% H2O, 20% CD3CN and 5% D2O at 298 K. Spin systems areshown in the TOCSY spectrum and the sequential connectivities in the NOESY spectrum. The one-letter code for amino acids as well as the residue number is used for the sequence assignments. Phe12has a NH chemical shift of 10.9 ppm and is not included in the diagram to improve visibility of theother peaks. Note that its downfield shift is a strong indicator of its hydrogen bonding.

Fig. 6. Structure of vhl-1. A, Stereoview of the 20 lowest energy structures of vhl-1 derived fromNMR restraint data, superimposed over the N, C, and Cα atoms of all 31 residues. The first and everytenth residue are numbered. B, Ribbon representation of the structure of vhl-1, showing its keyfeatures of a CCK motif and secondary structural elements.

Fig. 7. Hydrogen bonding interactions. A, pH titrations monitoring the changes in chemical shifts atbackbone amide protons. B and C, The cyclotide backbone and disulfide bonds are presented in line

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format, with the residues involved in hydrogen bonding interactions displayed in bold representation.Hydrogen bonding networks are highlighted with shaded circles.

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Table 2 Structural statistics for the family of 20 vhl-1 structures

Energies (kcal mol-1)Overall -982.33 ± 16.33

Bonds 6.99 ± 0.67

Angles 42.41 ± 4.16

Improper 6.16 ± 1.32

Van der Waals -67.60 ± 5.21

NOE 9.98 ± 2.01

cDih 0.65 ± 0.27

Dihedral 125.10 ± 7.26

Electrostatic -1106.22 ± 18.99

Atomic rms derivation (Å)Mean global backbone 0.32 ± 0.08

Mean global heavy 1.14 ± 0.14

Distance restraintsIntraresidue (i-j=0) 87

Sequential (|i-j|=1) 103

Medium range (|i-j|�5) 51

Long range (|i-j|>5) 78

Hydrogen bonds 20

Total 339

Dihedral angle restraintsφ 25

χ1 17

Total 42

Violations from experimentalrestraintsNOE violations exceeding 0.20 Å 0

diheral violations exceeding 3.0° 0

RamachandranMost favoured 75%

Additionally allowed 21.3%

Generously allowed 3.7%

Disallowed 0.0%

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Figure 1.

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Supplementary Table 1: Nanospray MS-MS Analysis of Vhl-1 Digested by Trypsinand Endoproteinase Glu-C

Ion type: b m/z z Ion type: y m/z zSC 191.0 1 E 148.1 1SCA 262.1 1 TE 249.1 1SCAM* 409.1 1 FTE 396.1 1SCAM*I 522.2 1 CFTE 499.2 1SCAM*IS 609.2 1 FCFTE 646.2 1SCAM*ISF 756.3 1 SFCFTE 733.2 1SCAM*ISFC 859.3 1 ISFCFTE 846.3 1SCAM*ISFCF 1006.3 1SCAM*ISFCFT 1107.4 1

Fragment 1m/z 627.7(Mass 1253.5)

SCAM*ISFCFTE 627.7 2E 148.1 1

VI 213.2 1 GE 205.1 1VIG 270.2 1 CGE 308.1 1VIGC 373.2 1 SCGE 395.1 1VIGCS 460.2 1 ISCGE 508.1 1VIGCSC 563.2 1 SISCGE 595.2 1VIGCSCK 691.2 1 NSISCGE 709.2 1VIGCSCKN 805.3 1VIGCSCKNK 933.3 1VIGCSCKNKV 1032.5 1

Fragment 2 - LNSISCGE 649.8 2 Fragment 2 - VIGC 874.3 2Fragment 2 - NSISCGE 706.3 2 Fragment 2 - VIG 925.8 2Fragment 2 - SISCGE 763.3 2 Fragment 2 - VI 954.4 2Fragment 2 - ISCGE 806.9 2 Fragment 2 - V 1010.9 2Fragment 2 - SCGE 863.4 2 Fragment 2 707.3 3Fragment 2 - CGE 906.9 2Fragment 2 - GE 958.4 2Fragment 2 - E 986.9 2

Fragment 2 §m/z 707.3(Mass 2118.9)

Fragment 2 707.3 3E 148.1 1

NK 243.1 1 GE 205.1 1NKV 342.2 1 CGE 308.1 1NKVC 445.2 1 SCGE 395.1 1NKVCY 608.3 1 ISCGE 508.2 1NKVCYL 721.3 1 SISCGE 595.2 1NKVCYLN 835.4 1 NSISCGE 709.3 1NKVCYLNS 922.4 1 LNSISCGE 822.3 1NKVCYLNSI 1035.5 1 YLNSISCGE 985.4 1NKVCYLNSIS 1122.5 1 CYLNSISCGE 1088.4 1NKVCYLNSISC 1225.5 1 VCYLNSISCGE 1187.4 1NKVCYLNSISCG 1282.5 1

Fragment 3m/z 715.3(Mass 1428.6)

NKVCYLNSISCGE 715.3 2

* Mono-oxidised Met§ Sequence of fragment 2: VIGCSCKNKVCYLNSISCGE

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Supplementary Figure 1: Surface representation of vhl-1.

A, showing the distribution of hydrophobic residues colored green, negatively chargedresidues colored red, positively charged residues colored dark blue, polar residues coloredlight grey, Met colored light blue and cysteine residues colored yellow. B, C and D, the viewsare rotated 90°, 180° and 270° about the vertical axis, respectively.

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and David J. CraikBin Chen, Michelle L. Colgrave, Norelle L. Daly, K. Johan Rosengren, Kirk R. Gustafson

structure and anti-HIV activity of VHL-1, A leaf-specific-expressed cyclotideIsolation and characterisation of novel cyclotides from viola hederaceae: Solution

published online April 11, 2005J. Biol. Chem. 

  10.1074/jbc.M501737200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2005/04/15/M501737200.DC1

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