523 2 His 2 -hairpin β 2009 European c ! Alberta, Edmonton, Alberta, Canada , orientation factor for energy transfer. 2 structures that confer structural | ,suggestingthatthehairpin-derived d K CFP, cyan fluorescent protein; FRET, fluorescence 2009 European Peptide Society and John Wiley & Sons, Ltd. c ! ry of this peptide with randomization of six residues with ay2009 PublishedonlineinWileyInterscience:23June2009 of Alberta, Edmonton, Alberta, Canada T6G2G2. E-mail: [email protected] Department of Chemistry, University of T6G 2G2 Present address: Gilead AlbertaCanada ULC, T6S1A1. 1021 Hayter Road, Edmonton, Alberta, Abbreviations used: resonanceenergy transfer;SPR,surfaceplasmon resonance;trpzip,tryptophanzipper; YFP, yellow fluorescent protein; Correspondence to: Robert E. Campbell, Department of Chemistry, University ∗ ‡ th submicromolar affinities. Interestingly, substitution of key to as ‘genericseparated protein the parts of scaffolds’: their tertiary proteinsstability that and have the effectively function parts [12]. that Aintended desirable confer for feature the intracellular of molecularwhich a applications recognition is generic is generally protein aallow thought scaffold relatively access to small to reduce size, epitopesdomains. that nonspecific Among are binding the sterically smallest and occluded validatedare generic from zinc protein fingers larger [13,14] scaffolds and the Trp-cage motif [15]residues, at respectively. just Despite their 26 small and size, 20 these peptides meet the basic criteria of aof generic the proteins structures scaffold because that thecoordination confer of parts a structural zinc ion stability in a zinc (i.e. finger andof the the aTrp-cage)areindependentof buried Cys tryptophan thepartthestructureusedforthemolecular recognition function (i.e. the five contiguous residues on tem to identify extended trpzip-type peptides with additional nded trpzip-type variants is the 24mer xxtz1-peptide with the an unstructured peptide ‘loop’ inserted into the turn region. in the turn region and therefore not an ideal peptide scaffold. ucted and panned against immobilized streptavidin. We have re in the xxtz1-peptide with the loop insertion, we did attempt ∗ einvestigatedengineeredversionsofthetrpzipclassof the loop region for molecular recognition. Copyright hage display; structured peptides and Robert E. Campbell ‡ type peptide as a molecularscaffold recognition An engineered tryptophan zipper- :523–532 Copyright 15 fluorescence; protein engineering; p 2009; The nonimmunoglobulin domain proteins best suited for conversionintomolecularrecognitiondomainshavebeenreferred J. Pept. Sci. or peptides to have moleculardependent recognition on functions the that formation of are a not disulfide bond [2,10,11]. of recombinant engineered antibodyexpressed fragments in [7] living that can cellsaffinity of be and the intact retain antibody from theAn alternative which approach binding they is to were engineer specificity nonimmunoglobin derived proteins [8,9]. and in the reducing environmentto of form the the cytosolstability multiple due [6]. disulfide to Much an bonds work inability that has been are invested critical in for the their development (as opposed tocircumvented therapeutic by applications), recombinant expression this ofheavy genes problem and encoding light can the chains of be the antibodythis approach in is the limited target by the cell. poor However, folding efficiency of antibodies tools. However, theiris use generally in limited by livelarge proteins the across cell the technical cell membrane. intracellular challenge For research of applications applications getting these biological molecular recognitionmolecular recognition applications. reagents can Theseby be traditional versatile readily immunization generated or either provide researchers hybridoma with high techniques specificity and [4,5] high affinity and research in the reducingas environment research ofAntibodies reagents are, a ostensibly, or the cell’s protein family potential interior of choice are therapeutic for most valued leads [1–3]. Introduction Proteins and peptides capable of specific molecular recognition applications in the reducing environment of the cytosol, w Zihao Cheng In an effort to develop a structured peptide scaffold that lacks a disulfide bond and is thus suitable for molecular recognition (www.interscience.com) DOI 10.1002/psc.1153 Research Article Received: 3 February 2009 Revised: 15 May 2009 Accepted: 22 M portion plays an important role in preorganization of Although NMR analysis provided noto direct use evidence this for construct structu asstreptavidin a resulted scaffold in for the identification phage of display peptideresidues of sequences in randomized wi the peptide hairpin-derived portion libraries. of Panning the of peptide the resulted in resulting a libraries 400-fold against decrease in stabilizing interactions. One of the mostsequence promising KAWTHDWTWNPATGKWTWLWRKNK. of A these phage exte display libra side chains directed towards onealso face explored of the the use hairpin was of constr xxtz1-peptide for the presentation of peptides. We have previously shown thatare even substantially most destabilized highly by folded the membersTo introduction of address of the this issue, trpzip mutations we class used (i.e. a the FRET-based live 16mer cell peptide screening HP5W4) sys Keywords: Peptide Society and John Wiley & Sons, Ltd. Supportinginformation may be found in the online version of this article p :523–532 -terminus N 15 a 2009; CHENG AND CAMPBELL J. Pept. Sci. -terminus. The ‘peptide’ is the C -peptide structure [24]. (C) Proposed xtz1 -peptide. -peptide has GAQ appended to its xtz1 xxtz1 KAWTHDWTWNPA-----TGKWTWLWRKNK KAWT--WTWNPA-----TGKWTW--RKNE KAWTHDWTWNPGSSGSSGGKWTWLWRKNK KK----WTWNPA-----TGKWTW----QE KAWTHDWTWNPGGGGGGGGKWTWLWRKNE KAWTHDWTWNPGGGGGGGGKWTWLWRKNK KK----WTWNPGGGGGGGGKWTW----QE KAWT--WTWNPGGGGGGGGKWTW--RKNE KAWTHDWTWNPA-----TGKWTWLWRKNE -hairpin (Figure 1) [16]. Accordingly, we reasoned that -sheet structure upon randomization of a subset of its -sheet (the stem), flanking an unstructured sequence β β β -hairpin with additional residues inserted into the turn -termini. The C β folds investigated in this work. (A) Flowchart outlining the relationshi -terminus and YFP appended to its A N -and face of the it might be possible to find aretain particularly the stable variant that would amino acids or even insertionin of the turn additional region. randomized residues region is andisplay intriguing of constrained minimalbeen peptides previously scaffold proposed [18,19]. for for anof Such the engineered two peptide a copies intracellular composed of structure anneuropeptide antiparallel head has activator dimerizing [20], peptide which presumably derived formsstranded from a two- N HP5W4 -peptide b 2009 European Peptide Society and John Wiley & Sons, Ltd. -peptide NA NA NA NA c -peptide NA ! ated in this work. (B) Schematic representation of the xxtz1/loop Copyright -terminus. C -peptide based on analogy with the experimentally determined structure of the -helix in a zinc finger and the seven solvent-exposed xxtz1 α Proteins and peptides investigated in the this work -hairpin peptides [16] are another promising candidate β Schematic representation of the overall strategy and specific peptide scaf Not applicable, construct was not made. The ‘protein’ is the indicated peptide sequence with CFP appended to its Trpzip b a xtz1xxtz0.5 xtz1 Protein nametz1 Peptide name HP5W4 Peptide sequence Table 1. xxtz1tz1/loop xxtz1 indicated sequence with few or no additional amino acids appended to its xtz1/loop xxtz0.5/loop xxtz1/loop and GT appended to its NA -hairpins in aqueous solution [16]. One of the most stable trpzip- β type peptides reported(Table to 1) date [17]. Much is offrom two a the pairs of fold 16mer tightly packed stability cross-strand known of Trp–Trp as these pairs on peptides one arises for a smallamino acids generic in length, soluble, protein monomeric, and form scaffold. highly stable They are only 16 to 20 residues of the Trp-cage).preorganized As structure will minimize with the entropic proteins, cost of a aadopting peptide highly the conformation folded necessary and for targetthereby binding lead and to may higher affinity reagents. www.interscience.com/journal/psc structure of the Figure 1. the face of an of the various peptides and peptide libraries investig

524 525 AG) is the EK Tris, pH 7.5. DH10B pre- M GGA GCT C BAD promoter. spectral charac- . All sequencing # ara E. coli AG) contains both a )thatannealstothe # in vitro is six consecutive histidine 6 erived from pBAD/His B (Invitro- TCT GCA G His -EK-CFP(1–230)-TSGAQ-peptide- 6 www.interscience.com/journal/psc -His ,where term Cwithshakingat225rpm.Thefollowing ◦ N term C -AGG CTT TAC ACT TTA TGC TTC CGG C-3 # -CCC TCG TGA CCA CCC TGA CCT GG-3 The system for imaging of the fluorescence of bacterial colonies # recognition sequence for the protease enterokinase.coding The the DNA TSGAQ en- amino acid sequence (ACT AGT GTSAE-YFP(5–238)- reactions were analyzed atBiology Service the Unit. University Protein samples ofterization for experiments Alberta were first Molecular dialyzedThe into 50 protein m referred tocitrine [28]. as The protein YFP referred is[29] to as the that CFP Ala206Lys has is a [27] beenimaging variant were of variant used purchased cerulean from in of Chroma Technology. prior studies [23]. All filtersFRET-based Library used Screening in The CFP–peptide–YFP librariesously were described vector expressed pZC1 [23] using d thegen) previ- which contains the arabinose-dependent fluorescence 96-well platereader equipped with monochromators (Tecan). Plasmid DNA was isolated and sequenced forthat those exhibited clones the highest FRET emission ratios. Protein Purification Atypicalproteinpurificationprocedurestartedwiththeinoc- ulation of 1arabinose l (0.2% LB w/v) with mediaviously a containing transformed single with ampicillin colony the (0.1 of expression mg/ml) plasmid. and Cultures were day, protein wasper extracted II from (Pierce) 1 ml and of emission each spectra culture acquired using using B- a Saphire2 chromophore region of the genefor encoding sequencing CFP. of The the primer displayed used portionwas of 5 phagemid pComb3ZC contains both a SpeI and athe SacI restriction GTSAE site. The DNA sequence encoding (GGT ACC polymerase were purchased from New Englandwith the Biolabs supplied and buffers. used Ligations were performedligase using T4 (Invitrogen) DNA according toThe the composition manufacturer’s of instructions. thewas peptide determined portions by of dyeDYEnamic all terminator ET FRET cycle kit constructs sequencing (Amersham(5 using Biosciences) the and a forward primer KpnI and a PstI restriction site.for Preparation FRET-based of screening the is peptide described libraries in(Supporting Supplementary Information). Methods has previously been describedPetri in dish, fluorescence some images detailthat were [23]. acquired correspond For in to three a480/40 CFP channels given nm), donor FRET (excitation (excitationand 436/20 YFP nm, 436/20 acceptor (excitation nm, emission 500/20 nm, emission emissionImage 535/30 Pro 535/30 nm). plus nm), An (Media Cybernetics) macro wasidentification used for of automatic colonies withinpixel digital images, intensities summing of inand the exporting each the of dataemission to ratio three a was channels spreadsheet.FRET calculated channel For for by by each the intensity each colony, dividing inthat the the the had colony, CFP the donor intensity channel. highest Colonies inin emission the ratios the YFP and the acceptorused highest channel brightness were to picked inoculate usingampicillin a LB (0.1 sterile mg/ml) tip liquid and and grown media arabinose overnight at (0.2% (5 37 ml) w/v). Cultures supplemented were with residues that facilitate metal affinity purification [30], and ThepZC1 vectorcanbeusedtoexpressgenesencodingthe proteinsof general structure - - β β in vivo -hairpin se- β colonies where screening method -peptide(Figure 1and 2009 European Peptide Society and John Wiley & Sons, Ltd. [24]. We now report our -hairpin structures might in vivo c ! β xtz1 tructure. Furthermore, peptide Escherichia coli HP5W4 -hairpin. Our strategy involved extending β -Pro-Gly sequence in place of the unstructured D 5352Copyright :523–532 15 peptideinanefforttoengineeradditionalstabilizing interaction relative to 2009; proteinthatharborsthe20mer π HP5W4 xtz1 In previous work, we had applied our We recently developed a method for rapid high-throughput TRYPTOPHAN ZIPPER AS A SCAFFOLD To investigate the potential of our mostfor promising use 24mer as peptide a generic peptide scaffold,based we screening have [26] used to phage explore display- thefide use bond-free of scaffold this for minimal the identification and of disul- peptide ligands. screening approach. We have furtherquence extended to the 24 residuesamino with acid combinations at exhaustive each new screening pair of cross-strand of residues. all possible Table 1), which has ancation– additional cross-strand lysine–tryptophan continued efforts to engineerinto additional these stabilizing trpzip-type interactions peptides through the use of our J. Pept. Sci. to the manufacturer’s protocol. Restriction enzymes and Taq DNA digestion were routinely purifiedcation using kit the QIAquick or PCR QIAEXpurifiedusing purifi- II GeneJetplasmidminiprepkit(Fermentas)according gel extraction kit (Qiagen). Plasmids were General Methods and Materials All synthetic DNAgratedDNATechnologies.PCRproductsandproductsofrestriction oligonucleotides were purchased from Inte- Materials and Methods compelling, evidence supporting the existence ofpeptide the structure stem–loop is inconclusive.and Gellman Indeed, [21] an provided evidence NMRhairpin that promoting study even with by a Lai strongly of randomized residues (the loop) [19]. Although this design is the sequence by two randomized residues inhairpin. each Iterative strand of library the screening resulted inthe the identification of arrangement that is more conducive to FRET. tothe interactions into this colonies using quantitativeColonies exhibiting image higher FRET ratios acquisition are picked and and theirDNA plasmid processing. isolated. DNAresponsible sequencing for shows bringing the the two peptide fluorescent sequence proteins into an orientation, which resultsefficiency in is more manifested as efficientintensity a that FRET. is simultaneously ratiometric measured for Higher change hundreds of bacterial in FRET fluorescence each colony expresses a singleCFP protein and construct a composed YFP ofHighly flanking a structured an and intermediary compacted peptideCFP peptide sequence and sequences [23]. YFP bring proteins the into closer proximity, or a more optimal screening of thousands ofare peptide highly sequences structured to influorescence find live imaging ones cells of that plates [23–25]. of This system relies on be particularly amenable to conversion into stem–loopby replacement structures of their turn sequencesrandomized with residues. longer sequences of peptide sequences that areportion more of the effective stem–loop at peptide s formingsequences that the adopt extremely stem stable neuropeptide head activator) can serve to tether together the ends of an intervening 18 residuethat, unstructured regardless of loop. whether It the neuropeptide occurred headretro to activator sequence) (or is us its able to self dimerize or not, there may exist other loop, the SKVILF stem structure is onlyCD moderately stable. study A recent byconclude Anderson that and tandem colleagues copies [22] of EFLIVKS led (the the retro authors sequence to of ) M lof 1ml µ × culture :523–532 15 E. coli 2009; ) were calculated s for protein bind- d d K K C. A streptavidin pre- NaCl, pH 7.4) as the ◦ M CHENG AND CAMPBELL l/min. To generate each µ J. Pept. Sci. g/ml tetracycline. After 2 h µ phagewereaddedtothebeads 11 10 HEPES, 150 m × M ) was added a buffered solution of strep- C, the VCSM13 helper phage was added. M ◦ -peptide was obtained with a Varian 600 MHz loffreshlypurifiedproteininHEP-EPbufferwas )refertoproteinsofthetypeCFP–peptide–YFP, µ xtz1 biotin in PBS and added to 2 ml prepared XL1-blue xtz1 M g/ml carbenicillin and 10 µ culture and incubate at room temperature for 15 min. Pre- The FRET-based determination of solution spectrometer as previously described [24]. Phage Library Panning and Enrichment Construction of the phage libraryMethods. is described All in Supplementary following panning established protocols [31]. and Enrichments ofbinding streptavidin- peptides enrichment displayed on phage were were performed byagainst panning streptavidin performed magnetic beads by (Invitrogen).streptavidin Briefly, magnetic 20 beads werew/v) first in PBS incubated for with 1 h. BSAunder Control identical panning conditions (3% with experiments beads were that performed had beenwithbiotin.Approximately5 preincubated and gently mixed for 1 h.PBS The buffer beads were containing washed with 0.5%weakly 10 Tween20 bound to phage. removewith The 0.1 unbound m and adherent phage were then eluted spectrum of ing to streptavidin was performedbased on platerreader a (Tecan). Safire2 Briefly, monochromator- toFRET a construct solution (50 of n each purified with BIAevaluation 4.1 (Biocorethe Inc.) data software to the package Langmuir 1 which : 1 fit binding model. Results The names of peptidesmarized and in proteins Table used 1. We innames use this (e.g. the work convention are that the sum- where short bolded the peptide is a specific sequence of between 16 and 24 assumes only that therestate is and 100% no binding in binding the final in (low the FRET) state. initial (high FRET) coated chip (SA chip, Biocore Inc.)with was HEP-EP used for buffer all measurements (10 m was recorded and plottedtration. as The a data function was of fit with streptavidin an concen- equilibrium binding model that running buffer atsensorgram, a 30 flow rateinjected. Equilibrium of dissociation 10 constants ( tavidin (Roche) to thefluorescence intensity desired at final 530 concentration. and The 480 nm ratio (excitation of at 430 n E. coli warmed SB medium waswith then add 25 to the infected of enrichment and amplification, five bacteriallibrary colonies from were each picked andreveal cultured. the DNA gene sequencing waspeptide sequence portion. used that to corresponded to the displayed Binding Analysis Peptide sequences selected by phage displayfrom were their PCR amplified purified phagemeids andplasmid inserted into for an appropriate productionCFP–peptide–YFP of as proteinsKinetic of described analysis of the inperformed with these a general Biacore Supplementary X proteins instrument structure at 25 binding Methods. to streptavidin was Kanamycin was added to the culture 2of h phage later, continued and as the described preparation above. After three to four rounds of incubation at 37 - C M is ◦ AG) term 2mg N calcu- HNMR ∼ 1 z AcTEV -peptide -peptide / m was used to NaCl, 2.7 m 2009 European Peptide Society and John Wiley & Sons, Ltd. xxtz1 C. The M ◦ O, with internal c , where 2 ! E. coli (3513.97 )wererecordedon xxtz1/loop term M C z C(10min,10000rpm) / -peptide, we used SPPS ◦ OinH 2 m -peptide). MALDI-TOF-MS Copyright using the primers that had calculated) for TFE concentrations between -peptide were purified by RP C, the supernatant was loaded z ◦ / M m xxtz1 xxtz1/loop Tris pH 7.5. Isolated proteins were at 280 nm was used to determine 1 xxtz1/loop M pH 6, 10% D HNMRspectraof and the protein product purified as − 1 4 xxtz1/loop Cwithshakingat225rpm,cooledto4 cm (3446.84 ◦ PO 1 2 z 50 mg )sites,respectively.Astopcodonfollows − / E. coli ∼ m -peptide. We previously reported a plasmid [23] NaH C. -peptide, respectively. A calculated extinction co- ◦ M xxtz1 imidazole (pH 7.5) according to the manufacturer’s peptide in a 1 mm path length cuvette on an Olis DSM phosphate, pH 7.4) and the cells were lysed by a single Opeak.Variousspectrawereacquiredatpeptidecon- M M M 2 µ -peptide and the -peptide and Owith0.1%TFA.Thefractioncontainingeachtargetpep- xxtz1/loop 2 -EK-CFP(1–230)-AcTEV-AQ-peptide-GT- 6 produce the that can be used to express proteins of the general structure and Kpn1 (GGT ACC obtained with a Varian 500 MHzof spectrometer the with H presaturation 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) reference] were with 40 tional) and FRET efficiencies werepreviously determined described by [23]. trypsinolysis Errors for as threewere of reported more as measurements standard deviation. CD spectra were obtained aQuantaMasterspectrofluorometer(PhotonTechnologyInterna- the protease substrate sequence QNLYFQG,resent and AQ the and amino GT rep- acids encoded by the Sac1 (GGA GCT C debris was pelleted by centrifugation at 4 passage through a French Press (Thermo Electron). Insoluble cell and 1 ml of Ni-NTAFollowing resin 1 (Qiagen) h was of gentle added mixing to at 4 the supernatant. Hiload 16/60 Superdex 75was prepgrade equilibrated gel with filtration 50 m column that protocol. Proteins were further purifiedmatography on system an AKTAdesign (Amersham chro- Biosciences) equipped with a 0% and 50%, and temperatures between 5 and 50 fragment [23]. To produce the on a ABI 433A peptide synthesizerxxtz1 (Applied Biosystems). Both the also been used forused error-prone to PCR. transform Thedescribed resulting above. plasmid Following purification, was thewithAcTEVprotease(Invitrogen)toreleasethepeptide-containing protein was treated the Kpn1 site.and This ligated plasmid with was thebeen digested similarly PCR with amplified digested from Sac1 DNA pZC1/ and fragment that Kpn1 had www.interscience.com/journal/psc KCl, 10 m grown overnight at 37 on ice, and harvestedcell by pellet centrifugation was (10 min, resuspended 5000 in rpm). PBS The buffer (137 m in buffer [10 m onto a 6 mlin polypropylene column, 250 m washed, and gravity eluted centrations between 0.1 and 2 m Recombinant expression of a fusion protein in Peptide Production 17 CD spectrometer (Olis). efficient of 33 450 M tide was lyophilized to provide a fluffy white solid (yield and stored at 4 concentrated with a Centricon centrifugal filter YM-30 (Millipore) His Spectroscopy Fluorescence spectra for all proteins (40 n concentrations. chromatography on a ProsphereAssociates, HP Inc.) using C18 a 300A linearin column gradient H (Alltech of increasing acetonitrile xxtz1 and showed molecular weightslated) of and 3446.62 3513.06

526 527 (Table 1). 20 b 20 .ThisGlu24Lys X xxtz0.5 (W/T/S/P/R) had indeed resulted xxtz0.5 . c osition exhibited the least in vivo ,theFRETefficiencyincreased xxtz1 -Hairpin Peptides was subjected to three iterative β 19 measurements confirmed that the xxtz1 www.interscience.com/journal/psc X to xxtz0.5 in vitro xtz1 adenine (A), guanine (G), cytosine (C), or thymine measurements of FRET efficiency for purified with E24K substitution = to was designated tz1 Sequences identified 6 In vitro X No higher FRET variants identified -hairpin peptides in which it was shown that diagonal interactions [32]. The 19th p xxtz0.5 β π 5 library. Specifically, at the 5th and 20thpreference for positions Trp there residues, was paired strong with either aresidue positively charged (Lys, Arg,there or was His) a orAsp). strong another These Trp. preference observed preferenceswork At for on are the a consistent 6th charged with position, residue previous (His or bias, with some apparent(Leu preference or for Val). a hydrophobicsamples residue of each of thethe amino proteins acid showed combination that of His, the5, Asp, sequence 6, Leu, with and 19, Trp at(data and positions not 20, shown). This respectively, proteinThe had was peptide designated the portion of highestrounds of FRET random mutagenesis by efficiency error-prone PCR andproteins libraries were of screened for high FRET efficiency. Sequencingvariant of with the the highest FRET at the endonly of the the third Glu24Lys round substitution. showed Presumably,the in two highest of FRET the variant rounds variant was of identical to Characterization of Extended As shown in Table 3, empirical screen for high YFP/CFP ratio in the identification ofIn proteins going from exhibiting highfrom FRET efficiency. 59% tothe 67.9% overall to length 84.5%.fluorescent of proteins It increases, polypeptide is the sequence FRETdecrease, important efficiency all between to is other the expected factors note to being two the that same. as For example, proteins cross-strand pairs ofcation– Trp with Lys or Arg engage in stabilizing 5 ET KP HW GP MP WR WR X KGVW WHQW HDLW WHTR WDLW WDQW HHLW -Hairpin (W/K/H/R/D/G) β esis by using the codon ‘NNK’ where N 8 24 T E 7 2009 European Peptide Society and John Wiley & Sons, Ltd. 23 W 17 17 6 N . c T -terminus, was expressed T ! X 22 C 16 5 . 16 K W W a 21 24 15 R 15 E K xxtz1 xxtz0.5 xxtz0.5 K xxtz0.5 xxtz1 20 8 23 14 -peptide (Table 1) [24], we created T 14 N 7 G G Library 22 W 13 13 6 K T T xtz1 T 21 5 12 12 R (W/K/H/R/D/G) X A A 4 4 20 T 11 T 11 (W/T/S/P/R) 3 X P 3 were subject to saturation mutagen P 19 W 19 10 W 10 X X’ 2 T 2 N N A 5352Copyright :523–532 A 9 18 9 18 1 1 W W W W 15 GorT. Peptide sequences identified by FRET-based libraries screening = 2009; and screened by ratiometric fluorescence imaging of -peptide). -terminus and YFP fused to the (Table 2). Using the numbering of the 24mer peptide (which xtz1 N This sequence was designated as Positions marked ‘ This sequence was designated as E. coli We constructed a second library in which the 6th and 19th a b c 4th Error-prone PCR on 2nd K 1st K Table 2. (T), and K 3rd Error-prone PCR on TRYPTOPHAN ZIPPER AS A SCAFFOLD J. Pept. Sci. FRET-based Screening of Libraries of Extended with no fluorescent proteins attached, we add(e.g. the suffix ‘-peptide’ residues in length. When refering to the isolated peptide portions of a seriestheir of amino variants acid that sequence exhibited than had a been stronger observed preference in in the first positions were randomized to20th positions all were amino randomized to acids acommonly and subset selected of the in the the 5th residues firstsecond most and library library for (Table high 2). FRET Screening variants of resulted this in the identification residue (Arg or His)(Met, paired Lys, with and Gly a observed) Trp, paired with or a seemingly Pro (Table any 2). residue for the eightsequenced the colonies section of exhibiting cDNAthe encoding the FRET the construct. peptide highest The portion sequences of at YFP/CFP showed the randomized a ratios, 5th modest and preference 20th positions for a positively charged the in colonies [23–25]. We screened several thousand colonies and, xtz1 will be used for the remaindernoted), of the the manuscript unless inserted otherwise residuesgenetic are library at of positions these 5, 400 6, peptide 19, variants, and 20. with A CFP fused to a library ofresidues, peptide one variants randomized in andinserted after one which the 4th threonine, two and were 16th additional residues genetically of pairs the peptide of portion of Peptides In an effort topreviously reported 20mer explore the possibility of further extending our - :523–532 and less, 15 M xxtz1/loop peptide was -peptide. (B) CD 2009; HNMRspectrum 1 xxtz1 CHENG AND CAMPBELL -peptide showed only a ), a characteristic that is xxtz1/loop- M J. Pept. Sci. es. (A) CD spectra of identical -peptide, and xxtz1/loop HP5W4, xtz1 HNMRspectroscopy(SupplementaryFigure1). 1 peptide concentration, the peaks in the NMR spectrum CD characterization of peptid -peptide (Supplementary Figure 1). Indeed, backbone HN M xtz1 The apparent lack of structure for the generally associated with[16]. a However, highly CD analysis folded of relatively trpzip-type weak structure exciton coupling bandband at at 230 approximately nm 205 and nm,loss consistent a of with negative the almost critical complete tryptophanrandom packing coil interactions character and (Figure increased 3). confirmed by line widths were decreasedaddition but of dispersion remained TFEfurther poor. decrease at The in concentrations lineof widths of and peaks. somewhat up Coupling betterthe constants dispersion to range for of 50% all 6–8peptide resulted Hz. HN spectrum The resonances in contrasts overall starkly fell appearanceof with of within the peaks the excellent observed dispersion inof the previously acquired resonances for residues that are present in both peptides are Figure 2. concentrations of loop sequence havepeptide sequence been more replacedits hydrophilic solubility. by and, Indeed, serinein potentially, the aqueous improve to solution purified (greater make peptide than was the 5 m highly soluble At 2 m time) and unstructured. At concentrations of 1 m were broad and poorly dispersed, indicatinglikely that aggregated the peptide (resulting was in an increased rotational correlation melting curves at 227 nm for the three peptides represented in (A). - C. 2 6 6 5 ◦ . . . . 0 0 0 0 xxtz1 ± ± ± ± xxtz1 (%) 6 4 3 3 FRET . . . . is highly series, as efficiency and 74 68 65 51 -peptide in xxtz1/loop xxtz1/loop Clowerthan 2009 European Peptide Society and John Wiley & Sons, Ltd. ◦ xxtz1 xxtz1 / c xxtz1 ! xxtz0.5 -peptide all exhibit -Hairpin Peptide xtz1 / β xxtz1 tz1 have FRET efficiencies of Copyright Protein insertion tz1/loop with loop xtz1/loop xxtz1/loop xtz1 xxtz0.5/loop -peptide (Figure 2A) confirmed 8% [25], respectively. Of course, . 0 and xtz1 (20 residues) have FRET efficiencies peptide was expressed and purified. ± -peptide, and 0 tz1 . and 7 7 . . xtz1 0 1 1 0 xxtz1- (Figure 2B). Close inspection of Figure 2B loop (refer to Table 1) diminished the FRET ± ± ± ± 7 (%) 5 1 9 0 FRET . . . . C) that differences in the peptide-fold stability efficiency HP5W4 84 70 67 59 ◦ HP5W4 HP5W4, xtz1 -peptide,Table 1).Theprimarydifferencebetweenthe -peptide and the peptide portion of the FRET efficiencies for proteins with and without loop 1% [23] and 44 provides qualitative support for the conclusion that the ± (16 residues) and 2 . xxtz1 tz1 To determine whether the peptide portion of To further investigate the peptide portion of the xxtz1 xxtz0.5 xtz1 Protein tz1 insertions Table 3. xxtz1/loop structured when removedprotein from fusion protein, the context of the fluorescent peptide portion of thistemperature protein as the is preceding at members of least the series. as structured at room of the peptide). The CD melting curve showed that the and 18. However, a slight red shiftthe indicates packing possible of differences in the indole moieties andindicates a new some minimum random at 205 coil nm conformation (possibly at the ends the CD spectra and the signalcomparison with magnitude of the that the additionalpacking of residues the tryptophan do indole side chains not at positions substantially 7, 9, disrupt 16, the positive band atare attributed approximately to 230packed nm exciton indole coupling [16]. sidechains of of These tryptophan the [33]. features two The pairs similarities of of closely AcharacteristicfeatureoftheCDspectraoftrpzip-typepeptidesis astrongnegativebandatapproximately215nmandastrong all other factors are notwe the same have in purposely the selectedthe for peptide highest sequences FRET that efficiency.for provide The high FRET efficiency measured proteinisthatfourofthesevenglycineresiduesintheunstructured xxtz1/loop peptide has a melting point approximately 15 It is only at temperaturestemperature substantially (20 higher than the screening variant, we synthesized and( purified a synthetic peptide analog 47% and 44%, respectively. A similarwas analog not of prepared, but its FRET44% efficiency due is to certain the increased to peptide be length. less than for an unstructured linker of the same length. As mentionedCFP–YFP above, fusion proteinslength with to the unstructured peptides linkers of identical in efficiency for allremained four substantially greater proteins. than However, that observed the (or FRET expected) efficiencies www.interscience.com/journal/psc with unstructured polypeptide linkers of identical lengths toof those of 47 As shown insequence Table with 3, a Gly replacement of the Ala-Thr of the turn manifest themselves. Insertion of an Unstructured Loop into a its precursor an effectively identical degree of melting between 5 and 20 showed that

528 529 to but and HPQ, library 2or3 . face E. coli xxtz1/EMQDHPQ xxtz1/EMQDHPQ xxtz1/EMQDHPQ for M .Forthesakeofcomparison, µ library, the peptide containing library and the relevant portion of in going from www.interscience.com/journal/psc loop d ,isidenticalto library. All five sequences (two were K face 8 and 130 indicates that the hairpin-derived portion ,respectively(SupplementaryFigure2). M 8 8 independent clones were obtained for the loop 8 10 xxtz1/EMQDHPQ libraries, respectively. Following several rounds of × sof320n d loop K ,producingthecodonCATthatencodeshistidine.An and 4 7 and 10 xxtz1/EMQDHPQ/Gly E. coli Theportionof theplasmidencoding thepeptidewassequenced It is well documented that peptides with a His-Pro-Gln (HPQ) × 10 constitute the remainder of the HPQ motif. for five coloniesenrichment obtained of after the three rounds of panning and face panning against streptavidin immobilized on magnetic beads,phage the output was observed toboth absolute dramatically number increase of in phage termsto eluted of a as control well as (Figure 4). outputidentically relative The to control the experiments selection were experiments,streptavidin performed was except preincubated the with immobilized an excess of biotin. sequence bind to streptavidinselected with from high peptide affinity libraries and pannedAs tend against designed, to streptavidin this be [34]. sequenceas was no not more presentto in than construct the two the consecutive library.protocol would We result residues in had the were discovery anticipatedthat, of randomized perhaps, that a mimicked novel the structured the motif HPQ selection biotin-binding site. motif After in four rounds its of ability selection,picked five to from clones were block the enriched the the plasmids sequenced. As shownthe in same Table 5, peptide all sequence fiveHPQ sequence. which, clones Apparently, had interestingly, a single contained Afor the to Asn C 8 mutation in (AAT) the mustin codon have been introduced duringintentional amplification Pro at position 9 and a Gln at the randomized position of the putative stem–loopthe motif has HPQ a key role motifinspection in pre-organizing for of binding availablebetween to streptavidin x-ray and streptavidin. crystalunlikely HPQ-containing On that peptides, structures the it the of relativelycould appears basis be distant involved complexes in Trp of specific binding residues interactions[35]. with of It the has protein the previously peptides beenrandomized shown that peptides phage cyclized display with panning disulfide of bonds results in the with the residues at positions 3,with 5, Gly. 7, 9, SPR 16, analysis 18, of 20, bothshowed and proteins 22 under replaced identical conditions 7 where one of thefour additional unique amino sequences acids use (X) onlyfor is one Pro, Asp of (Table the suggesting 5). two thatrearrangement All possible of they a codons single are progenitor sequence sister in clones that arose from This 400-fold decrease in identical) contained theunique HPQ sequences sequence followed the and sequence three pattern of of EX the four xxtz1/EMQDHPQ/Gly xxtz1/EMQDHPQ/Gly Characterization of Streptavidin-binding Peptides To quantitativelypeptides assess selected from the the streptavidin-binding affinity of the EMQDHPQ sequence wascomplete further peptide characterized sequence by was SPR. clonedgenetically from This inserted the phagemid between and CFPstem–loop and YFP structure toentity as help stabilize well to the aswas increase designated to the createaproteincontaininga‘linearized’versionofthispeptide SPR a larger response.sequence molecular The was also resulting prepared.as The protein linearized version, designated - β xtz1- -peptide -peptide -peptide a xtz1 --KNE -peptide than in X W X xxtz1/loop xxtz1/loop -hairpins as scaffolds β 2009 European Peptide Society and John Wiley & Sons, Ltd. GKW GGKWTWLWRKNK X c ! -peptide spectra acquired xxtz1/loop -peptide. Experimental conditions ------XXXXXXX X xxtz/loop WNP X -Hairpin Scaffold and a Loop Insertion -peptide were constructed and screened. xxtz1/loop W β X were subject to saturation mutagenesis by using X 5352Copyright :523–532 -peptide that align with Trp 7, 9, 16, and 18 of KAW-- KAWTHDWTWNPG 15 xxtz1/loop xtz1 -peptide resonate at chemical shifts of 9.17, 8.78, 8.70, Phage display libraries 2009; CD spectrum of library was generated by replacing the GSSGSSG loop with library was constructed by genetically randomizing six amino -peptide. For example, the HN hydrogen atoms of the Trp Positions marked a Table 4. Library nameFace Sequence of peptide portion the codon ‘NNK’. Loop TRYPTOPHAN ZIPPER AS A SCAFFOLD To explore the potential of trpzip-type Phage Display with a Scaffold at any temperatureresults indicate with that this or peptide doesstructure without not to fold any into TFE a appreciable hairpin-type (up degreeNMR experiment. under to the conditions 50%). of These the xxtz1/loop and 8.73 ppm, respectively. Resonanceswere at similar not chemical shifts observed in the are identical to those used in Figure 2A. J. Pept. Sci. acids that have side chains oriented towards the face of the the pIII protein of the M13panned Ff with filamentous selection bacteriophage for [31] streptavidin and binding.face The Although the apparent lack of structure in the would seem to make it a poor candidatethe for this purpose, FRET we found resultspeptide intriguing libraries enough were displayed to as fusions justify to the a truncated attempt. version The for molecular recognition, phage displaypeptide libraries and based on shifted substantially more upfield in xtz1 residues of Figure 3. flanked by single Gly residues on either side (Table 4). Libraries of introduction of degenerate NNK codons. The loop asequenceofsevenrandomizedresidues(usingNNKcodons) hairpin that is opposedchains to are packed. the Specifically, face the11, 15, where residues and 17 at the (Figure 1 positions tryptophan and 4, Table side 4) 6, were randomized 10, through the , - d K xtz1 at strep- :523–532 8 15 2009; -hairpin peptides. -hairpin structure, β β CHENG AND CAMPBELL values of 100, 50, 80, and d J. Pept. Sci. K , the highest concentration M µ xxtz1/EMQDHPQ/Gly 5 . obtained by FRET and SPR analysis of -peptide. A substantial amount of time M -peptide has been further extended to , respectively. No significant change in the library to streptavidin. (C) Phage output for each xxtz1 xtz1 are in reasonable agreement. face -hairpin peptides to identify those that are highly β xxtz1/EMQDHPQ, xxtz1/EMDHPQN, xxtz1/ELDNHPQ for xxtz1/EMSDHPQ M tive DNA sequencing data for the randomized portion of the peptide and emission ratio was observed for xxtz1/EMQDHPQ tested. Given the differences in experimental protocols, the peptide [24] from thecurrent highly work, folded the 16merproduce the HP5W4 24mer [17]. In the at concentrations of streptavidin well belowfusion the protein, concentration might of be attributablecrowded to molecular orientation environment of effects the in multiple protein the Regardless complex. of direction of the FRET change, the datawith was a consistent two-state model in which distinct YFP/CFPs ratiosciated were with asso- both the bound and unbound states.to Fitting an these equilibrium data binding model gave 90 n values of 100 and 320 n Discussion FRET-based Screen for Identification of Structured Peptides In the pursuitmolecular recognition applications of in the reducing environment a of the cytosol, constrained we explored peptide the use of scaffold trpzip-type suitable for tavidin concentrations up to 1 the introduction of multipleserve randomized as amino the acids basispeptides and for could thus libraries be from selectedmind, which by we high developed phage affinity binding a display.libraries FRET-based With of strategy this for aimfolded screening in in large thepreviously cytosol used of to a live guide bacterial the cell. engineering This of strategy the was 20mer We reasoned thatpossibly an engineered exceptionally by stable interactions into introduction an of already very additional stable stabilizing structure, may tolerate as de- 1 1 1 2 5 library, # of occurrences loop 2009 European Peptide Society and John Wiley & Sons, Ltd. sintherangeof c ! d K --KNE [37]. N a W M F µ xxtz1/EMQDHPQ Copyright GKW library to streptavidin. S loop library, we exploited a FRET-based ------Q WHP loop wereeachincubatedwithvarying concen- Q sequence of peptide portion W 8 P E M S D H P Q E L D N H P Q E M D H P Q N E M Q D H P Q gag atg tcg gat cat ccg cag gag ctg gat aat cat ccg cag gag atg gat cat ccg cag aat gag atg cag gat cat ccg cag s in the range of 100s of d KAW-- library shown). (B) Phage output for each round of selection for binding of the K in Table 4) are provided. Peptide sequences obtained from panning against [36]. It has also been shown that linear HPQ-containing loop M Construction and panning of phage display libraries. (A) Representa face loop library name Randomized residue positions are in boldface type and the To further characterize the solution binding affinities of pep- XXXXXXX a Table 5. streptavidin the nucleotide and amino( acid sequences of only the loop portion streptavidin-binding motif HPQ is underlined. For the 100s of n xxtz1/EMQDHPQ/Gly (Table 5) werevector cloned to make into constructs analogous thescribed to in the preceding CFP–peptide–YFP paragraph. These four proteins along with expression tides selected from the binding assay. The remaining three unique peptide sequences www.interscience.com/journal/psc identification of HPQ-containing peptides with Figure 4. sequence ( round of selection for binding of the creased (Supplementary Figure 2). This decrease in FRET efficiency, streptavidin would result in increasedclose FRET proximity of efficiency multiple due acceptors to forratio each the donor, was the observed YFP/CFP to decrease as streptavidin concentration in- trations of streptavidin and their YFP/CFP ratio determinedconcentration. at Although each we had expected that binding ofcopies the of four the CFP–peptide–YFP fusion protein to the tetrameric peptides have

530 531 the versus -hairpin are β -hairpin peptides can β play of these noncovalently con- www.interscience.com/journal/psc -hairpin scaffolds may one day be useful β -peptides are unfolded when in isolation, but xxtz1/loop It is as yet unclear to us how the hairpin-derived portion of these Ingenuity and was madeby possible the with UniversityInnovation. infrastructure of We provided thank Alberta Cold and Spring Harborin the for offering Phage Canadian their course Display Foundationfor of for providing Proteins the phage and display vector,critical Peptides, reading and of this Jamie Carlos manuscript. K. The authors Scott F. also thank forCartmell, Jonathan Barbas her Svetlana III Sapelnikova,of Alberta Wayne MBSU, Moffat, andlaboratory, Mark for the technical Miskolzie assistance. University of RECChair holds the in a Bioanalytical NMR Canada Chemistry. Research spectroscopy In this work, we have demonstrated that noncyclized promising candidates for use asbut further molecular engineering recognition will be scaffolds, required beforeerally they useful become gen- for intracellularWe molecular found recognition that when applications. fusedsuch to as a a large fluorescent solubilizing protein protein orvalent partner a interstrand phage interactions coat protein, of theact certain nonco- as a surrogatestructure. Furthermore, phage for dis cyclization instrained motifs a can result stem–loop in thewith identification type affinities of that binding peptide are peptides comparable with thosetional achieved disulfide-cyclized with peptides. tradi- Peptide ligands basedor on related trpzip-derived these for intracellular molecular recognition applications. Althoughdisappointing that it the is peptide structures investigated in thiswere work grossly destabilized by thethere insertion may of unstructured exist loops, otherfolded ‘stem–loop’ peptide structures sequences when free that in solution. can form highly Supporting information Supporting information may be found in thearticle. online version of this Acknowledgements This research was supported by grants from NSERC and Alberta Conclusion are structurally stabilized whensuch fused as to a a large fluorescentample protein protein precedent partner or for a suchfor phage behavior, binding as coat peptides differences protein. genetically in Therefree fused binding properties is peptides with have a been reported protein forpeptide/protein a combinations variety [39–42]. of different binding properties (investigated by both SPRbasedsolutionassay),theselooppeptidesseemtobehavesimilarly analysis and a FRET- to previously reportedstreptavidin. covalently Specifically, the affinities cyclized obtained for the peptidetype stem–loop structures ligands were on par for with affinitiescyclized obtained peptide libraries with [36]. disulfide- Furthermore, linearizing the peptide by removing sidestability chains that of are thedecrease normally hairpin-derived in critical binding portion for affinity. Thesesupport the resulted characteristics for fold provide in the strong conclusion akey that factor 400-fold in the dictating hairpin-derived the binding portion affinity. is a peptides could be having such a pronouncedwith effect which the on loop the portion affinity binds to streptavidin. Onethat possibility is the .This in vitro -hairpin -peptide β HP5W4 xxtz1 -hairpin peptides. screen of selected β -hairpin peptides [38], 2009 European Peptide Society and John Wiley & Sons, Ltd. β c in vitro ! 2/3). The important point is -peptide and -Hairpin Scaffolds -hairpin or as a loop inserted at β > β xtz1 2 κ nging the two fluorescent proteins 5352Copyright :523–532 15 -hairpin peptides as scaffolds for use in phage β 2009; -hairpin peptides has decreased the relevance of these latter β It is now evident that the FRET-based screening strategy is An apparent limitation of the FRET-based screening strategy is In this work, we explored the ability of trpzip-type TRYPTOPHAN ZIPPER AS A SCAFFOLD J. Pept. Sci. arelativeorientationthatisfavorabletoFRETduetothe dipole–dipole alignment (i.e. peptide may simply adoptrandomly oriented a fluorescent conformation proteins into that closerthird proximity. brings The mechanism the is two conformation that the that peptide biases could the adopt attached a fluorescent structured proteins to is, the folding/unfolding equilibriumfolded structure, may effectively lie bri moreinto towards closer the average proximity. The second mechanism is that the are three possiblesequence mechanisms might produce by higher which FRETgiven efficiency. a a The particular first particular folded ispeptides peptide that, conformation, may a higher exist fraction in of the the folded state at a given time. That temperature. Highly structured peptidesFRET provide between more two efficient structured fused (or fluorescent unstructured) proteins peptides than of do similar poorly length. There this screening strategy, and we areits now effectiveness in and a limitations. position to reflect on effective for identifying peptides that are highly structured at room and effort has been spent in developing [23] and exploiting [24,25] In contrast to the fairlycarried extensive out amount on of phage workrelevance display that to of has this cyclized been work, disulfide-cyclized peptides, and, of most FRET constructs by measuringtemperature. the YFP/CFP ratio at an elevated Phage Display with Noncyclized stability could be sacrificedthat to is adopt a amenable structuredmay to conformation be higher useful FRET to conduct efficiency. a For secondary future work, it equivalent or decreased thermal stability relativecan to be rationalized in termsmentioned of the second above, and third which mechanisms improvement do in not thermal imply stability. or Indeed, require the overall an thermal overall is based on theproduce fact substantially that both higherof FRET a efficiencies CFP–YFP in fusion the protein, context but the isolated peptides have to explain a higher FRET efficiency. that peptides that are selected for highdo FRET not necessarily at have room improved thermal temperature stability. This conclusion that all three factors require that the peptide be highly structured peptides isolated from the loop library. With respect to their theturnregion.Althoughbothpresentationformatsresultedinthe identification of streptavidin-binding peptides containing the HPQ consensus, we have focussed our attention on characterizing the two concerns. peptides for the display ofon polypeptide the libraries highly structured presented face either of a Agrowingdemandforresearchtoolsforuseinthereducing environment of the cytosol coupled with thetype advent of the trpzip- discrepancy including: thecyclized peptides, relative a narrower ease rangeof of of a applications disulfide in forming bond which would lack disulfide- recently) be a advantageous, and lack (until of relatively highly stable candidate relatively little effortnoncyclized has been investeddisplay. in the There develpment are on likely several contributing reasons for this : : : : : 22 34 99 338 249 2008; Protein 2004; 1995; 2005; :523–532 2004; 1990; Biochemistry Glycobiology :1321–1325. 15 FASEB J. 6 :29188–29194. 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