Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.A combination of classical site-directed mutagenesis, genetic code engineering and bioorthogonal reactions delivered a chemically modified barstar protein with one or four carbohydrates installed at specific residues. These protein conjugates were employed in multivalent binding studies, which support the use of proteins as structurally defined scaffolds for the presentation of multivalent ligands.DFG, SFB 765, Multivalenz als chemisches Organisations- und Wirkprinzip: Neue Architekturen, Funktionen und Anwendunge

Artner, Lukas M.

Beceren-Braun, Figen

Bohlke, Nina

Budisa, Nediljko

Dernedde, Jens

Hackenberger, Christian P. R.

Merkel, Lars

Weise, Christoph

Chemical Communications

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 This article is part of the  Glycochemistry & glycobiology web themed issue   Guest Editors are Professor Peter H. Seeberger and Dr Daniel B. Werz   Please visit the website to access the other articles in this issue:-http://www.rsc.org/chemcomm/glycochemistry       Published on 09 November 2011. Downloaded by TU Berlin - Universitaetsbibl on 30/03/2016 13:20:36. View Article Online / Journal Homepage / Table of Contents for this issue522 Chem. Commun., 2012, 48, 522–524 This journal is c The Royal Society of Chemistry 2012Cite this: Chem. Commun., 2012, 48, 522–524Site-selective modiﬁcation of proteins for the synthesis of structurallydeﬁned multivalent scaﬀoldswzLukas M. Artner,a Lars Merkel,b Nina Bohlke,b Figen Beceren-Braun,c Christoph Weise,aJens Dernedde,c Nediljko Budisa*b and Christian P. R. Hackenberger*aReceived 28th September 2011, Accepted 17th October 2011DOI: 10.1039/c1cc16039gA combination of classical site-directed mutagenesis, geneticcode engineering and bioorthogonal reactions delivered a chemicallymodiﬁed barstar protein with one or four carbohydrates installedat speciﬁc residues. These protein conjugates were employed inmultivalent binding studies, which support the use of proteins asstructurally deﬁned scaﬀolds for the presentation of multivalent ligands.Post-translational protein modiﬁcations play an important rolein the regulation and organization of biological processes ofliving organisms and are therefore of common scientiﬁc interest.1In mammalians, one of the most frequent and complexmodiﬁcation processes is protein glycosylation, which resultsin the attachment of oligosaccharides as N- or as O-linkedglycans.2 Glycoproteins play a major role in recognitionevents, such as cell–cell interactions, and protein–antigenrecognition.3 Binding events between glycosylated cellularsurfaces and carbohydrate-recognizing proteins4–namely lectins–often occur in a multivalent or cooperative fashion;5 a mechanismthat is also used by pathogens for infection.6 Interestingly, analogousmonovalent carbohydrates usually only bind to lectins in thelow millimolar range.7 Consequently, investigations focussingon the understanding of multivalent interactions as well as onthe design of artiﬁcial multivalent binding systems have recentlyattracted considerable attention, in particular because carbo-hydrates and carbohydrate-recognizing proteins are consideredas attractive medicinal targets in cancer research.8 Over the lastfew years, numerous groups have explored the multivalency eﬀectbetween carbohydrate-presenting scaﬀolds and their receptors.Commonly utilized scaﬀolds include polymers,9 dextrins,10nanotubes11 and nanoparticles.12 Although the chemical accessand functionalization of many polymeric systems is welldeveloped, they often lack information about the exact numberand the structural presentation of the multivalent ligands. Forother scaﬀolds that have addressed these issues, includingfullerenes13 or viral capsids,14 changing the rigidity and ﬂexibilityof the scaﬀold is often limited.In this communication, we describe a systematic and modularway for the generation of multivalent binding systems by usingproteins themselves as structured scaﬀolds, which present adeﬁned number of carbohydrate ligands. This can be achievedby employing unnatural protein translation for the ribosomalincorporation of a speciﬁc number of unnatural functionalgroups into a protein, which can be chemoselectively conjugatedafter expression.15,16 In particular, we used homopropargylglycine(Hpg, Scheme 1) as a non-canonical amino acid16 in the supple-mentation incorporation method (SPI), which is based on in vivosense codon reassignment. The use of an auxotrophic bacterialhost along with a controlled protein expression allows the residue-speciﬁc replacement of a particular canonical amino acid by anon-canonical one.2,15–17 It is important to note that thismodular concept allows the positioning of multivalent ligandsat preselected sites within various protein structures as well as astraightforward variation of the linker length between thescaﬀold and the ligands.18In our model study, we attempted to engineer an artiﬁciallyglycosylated protein for lectin binding studies. We chose thestructurally well-deﬁned cysteine-free ‘‘pseudo-wild-type barstar’’c-b* from Bacillus amyloliquefaciens as the protein scaﬀold.16,19c-b* is a 10 kDa protein composed of 90 amino acids with onlyone methionine residue.19 The 3D-structure of parent c-b*20revealed three solvent-exposed positions (K23, E47 and K79)Scheme 1 The structure of c-b* from Bacillus amyloliquefaciens withmutations (K23M, E47M and K79M) indicated for c-b*4M.Methioninewas subsequently globally replaced by Hpg during protein expression.a Freie Universita¨t Berlin, Institut fu¨r Chemie und Biochemie,Takustr. 3, 14195 Berlin, Germany.E-mail: hackenbe@chemie.fu-berlin.de; Fax: +49 30 838 52551b TU Berlin, Institut fu¨r Chemie, Franklinstraße 29, D-10587 Berlin,Germany. E-mail: budisa@biocat.tu-berlin.de;Fax: +49 30 314 28 279c Charite´—Universita¨tsmedizin Berlin, Institut fu¨rLaboratoriumsmedizin, Klinische Chemie und Pathobiochemie, CBF,Hindenburgdamm 30, 12200 Berlinw This article is part of theChemComm ‘Glycochemistry and glycobiology’web themed issue.z Electronic supplementary information (ESI) available: Brief outlineof the synthesis, MALDI-spectra. See DOI: 10.1039/c1cc16039gChemComm Dynamic Article Linkswww.rsc.org/chemcomm COMMUNICATIONPublished on 09 November 2011. Downloaded by TU Berlin - Universitaetsbibl on 30/03/2016 13:20:36. View Article OnlineThis journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 522–524 523which were subsequently exchanged to methionine via site-speciﬁc mutagenesis, giving rise to c-b*4M (Scheme 1). In thisway, four alkyne-containing Hpg amino acids were eﬃcientlyintroduced to c-b*4M by SPI yielding the congener denoted byc-b*4M[Hpg], which can be used to conjugate carbohydrates tobarstar to four unnatural residues.16,21 In addition, c-b*1M[Hpg]was expressed containing a single Hpg at the N-terminus forcomparative lectin binding studies with mono-glycosylated barstarproteins.In our studies, we chose the well-established copper-catalyzed1,3-dipolar azide–alkyne cycloaddition (CuAAC),22 commonlytermed as ‘‘click’’ reaction,23 in particular because of the straight-forward accessibility of diﬀerent azide-functionalized carbo-hydrates24 with three linker lengths as well as the high expressionrates of proteins with alkyne-bearing amino acids.15,25For lectin binding studies of the glycosylated protein scaﬀolds,peanut agglutinin (PNA) was chosen, for which weak inhibitionwith galactose and stronger inhibition with lactose are known.Consequently, six b-linked azido derivatives of galactose (1–3)and lactose (4–6) with diﬀerent linker lengths were probed, whichwere synthesized from known protocols (see ESIz).9–12,24 TheCuAAC-reaction of c-b*4M[Hpg] with azido-sugars 1–6 wasperformed under optimized conditions recently reported by Finnand coworkers, using tris-(hydroxypropyltriazolylmethyl)amine(THPTA) as a Cu(I) stabilizing ligand.26 Non-commerciallyavailable THPTA was synthesized in 65% yield by usingCu(MeCN)4PF6 as catalyst. Strict implementation of thepreviously mentioned CuAAC protocol did not lead to fullyfunctionalized protein, but with an extended reaction time andlower temperature we managed to get full functionalization ofc-b*4M[Hpg] with all six azido-sugars to yield protein conjugatesb*1–b*6 (Scheme 2A), as veriﬁed by the corresponding gelshifts in gel electrophoresis andMALDI-ToFMS (see Scheme 2Band ESIz). Additionally, the single alkyne-containing c-b*1M[Hpg]showed full conversion with galactose 2 and lactose 5 to mono-glycosylated b*2[1] and b*5[1] (see ESIz).After puriﬁcation by dialysis, the inhibitory eﬀect of theartiﬁcial glycoproteins was probed via a competitive surfaceplasmon resonance (SPR) binding assay, where the inhibitionof sugar-functionalized barstar proteins on PNA binding tothe immobilized Thomsen–Friedenreich (TF) antigen wasanalyzed. In brief, the TF antigen coupled multivalently to apolyacrylamide backbone was coated to a Biacore chip surface.Binding signals of the analytes passed over the chip wererecorded as resonance units (RU). The respective RU forPNA binding alone was set to 100% and served as positivecontrol. Potential inhibitory compounds preincubatedwith PNA should then demonstrate reduced binding (X% ofcontrol). Measurements were performed with unfunctionalizedc-b*4M[Hpg] as control as well as quadruple functionalizedbarstar proteins b*1–b*6 and the mono-functionalized proteinsb*2[1] and b*5[1] (Fig. 1A). Unspeciﬁc inhibition of PNA byc-b*4M[Hpg] appeared to be very low (approximately 0.2%).In addition, it was found that the lactose conjugated proteinsb*4, b*5 and b*6 inhibited PNA-binding between 21% and44%, in which the ethyl-spaced lactose conjugate b*5 showedthe strongest inhibitory eﬀect of all proteins (Fig. 1A). Asexpected, the inhibitory eﬀect was signiﬁcantly lower forgalactose conjugates b*1, b*2 and b*3, which can be rationalizedby the previously mentioned lower carbohydrate speciﬁcity ofPNA. Finally, both mono-glycosylated proteins b*2[1] andb*5[1] showed a signiﬁcantly reduced inhibitory potency,thereby pointing towards increased binding due to the presentationof several lactose ligands. Free ligands in contrast showed norelevant inhibition at all (ESIz).In the next step, we focussed on the structural analysis of thelactose scaﬀolds, which showed the strongest inhibitory eﬀect.Speciﬁcally, we measured the ﬂuorescence of the parent proteinand its conjugates as changes in the emission spectra andquantum yields represent a speciﬁc ﬁngerprint of the proteintertiary structure.27 Barstar contains two solvent-exposed Trpresidues (positions 38 and 44) and buried Trp53.19 Trp-residuesnot only dominate its absorbance and ﬂuorescence proﬁles butalso play a crucial role in c-b* structural integrity.28 In c-b*Fig. 1 (A) Relative inhibition of PNA binding measured by SPR incompetition with c-b*4M[Hpg] (denoted as b*) and protein conjugatesb*1–b*6, b*2[1] and b*5[1]. The SPR data for the conjugates werenormalized against the unfunctionalized protein c-b*4M[Hpg];(B) ﬂuorescence emission spectra of c-b*4M[Hpg] (denoted as b*)and multivalent lactose glycoprotein conjugates b*4–b*6 and b*5[1]excited at 280 nm. For experimental details see ESI.zScheme 2 (A) Functionalization of c-b*4M[Hpg] with azido-sugars 1–6by CuAAC. (B) Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE; 12%) of parent c-b*4M[Met] (left) along with c-b*4M[Hpg](second from left) and related protein conjugates b*1–b*6. Expectedly,migration times of glycoprotein conjugates are shifted when comparedwith non-conjugated species. For experimental details see ESI.zPublished on 09 November 2011. Downloaded by TU Berlin - Universitaetsbibl on 30/03/2016 13:20:36. View Article Online524 Chem. Commun., 2012, 48, 522–524 This journal is c The Royal Society of Chemistry 2012a network of cooperative interactions exists around Trp53, whichis sandwiched between Phe56 and Phe74.19 These residues play acentral role in maintaining the stability of barstar and all thosefeatures are reﬂected in the ﬂuorescence emission maxima.As shown in Fig. 1B the ﬂuorescence emission spectrum ofc-b*4M[Hpg] is composed of a typical spectral shoulderbetween 330–340 nm (contribution of buried Trp53) and apeak maximum B350 nm (from solvent-exposed residuesTrp38/Trp44). The proﬁles of its glycoconjugates b*4–b*6and b*5[1] are essentially identical. Therefore, it can be reasonablyassumed that the structural integrity is not signiﬁcantlycompromised by the conjugation reaction, as no hypsochromicor bathochromic shift of the ﬂuorescence maximum could beobserved although the shoulder in the b*5[1] spectrum is slightlymore pronounced.In summary, we have presented a strategy for engineeringan artiﬁcial protein scaﬀold for multivalent binding studies byconjugation with carbohydrate moieties. We demonstratedthis by introducing one or four galactose and lactose residuesat preselected solvent-exposed sites in a barstar protein by acombination of unnatural protein translation and bioorthogonalfunctionalization. The SPR experiments show that this artiﬁcialprotein scaﬀold acquired the highest capacity to inhibit PNAbinding upon conjugation with four ethylene-glyco spacedlactose residues as opposed to the mono-glycosylated barstarprotein without any detrimental eﬀect on native tertiarystructure. We anticipate that the high-resolution crystal andsolution structures available for many proteins will oﬀeralmost ideal platforms for decorating those biopolymers withmultivalent ligands, including carbohydrate or even peptidemotifs. This will include proteins with diﬀerent structuralfeatures and various levels of rigidity and will provide a verypromising route for the design of other protein-based multi-valent systems. We believe that the strategy presented hereprovides a solid basis for the further development and designof molecular systems for multivalent binding studies.The authors acknowledge ﬁnancial support from the GermanScience Foundation (Emmy-Noether program, HA 4468/2-1),the SFB 765, the Boehringer-Ingelheim Foundation and theFonds der chemischen Industrie (FCI).Notes and references1 C. T. Walsh, S. Garneau-Tsodikova and G. J. Gatto, Jr, Angew.Chem., Int. Ed., 2005, 44, 7342.2 D. P. Gamblin, E. M. Scanlan and B. G. Davis, Chem. Rev., 2009,109, 131.3 D. A. 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Site-selective modification of proteins for the synthesis of structurally defined multivalent scaffolds

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Site-selective modification of proteins for the synthesis of structurally defined multivalent scaffolds

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