Analytica Chimica Acta 2003 (497) 101

Analytica Chimica Acta 497 (2003) 101–114

Heterobifunctional crosslinkers for tethering singleligand molecules to scanning probes

Christian K. Rienera, Ferry Kienbergera, Christoph D. Hahna,Gerhard M. Buchingera, Innocent O.C. Egwima, Thomas Haselgrüblera,

Andreas Ebnera, Christoph Romanina, Christian Klampflb, Bernd Lacknerb,Heino Prinzc, Dieter Blaasd, Peter Hinterdorfera, Hermann J. Grubera,∗

a Institute of Biophysics, J. Kepler University, Altenberger Str. 69, Linz A-4040, Austriab Institute of Chemistry, J. Kepler University, Altenberger Str. 69, Linz A-4040, Austria

c Max-Planck Institut für Molekulare Physiologie, Otto-Hahn-Str. 11, Dortmund D-44227, Germanyd Institute of Medical Biochemistry, Vienna Biocenter, Dr. Bohr Gasse 9/3, University of Vienna, Vienna A-1030, Austria

Received 5 June 2003; received in revised form 30 July 2003; accepted 15 August 2003

Abstract

Single molecule recognition force microscopy (SMRFM) is a versatile atomic force microscopy (AFM) method to probespecific interactions of cognitive molecules on the single molecule level. It allows insights to be gained into interactionpotentials and kinetic barriers and is capable of mapping interaction sites with nm positional accuracy. These applicationsrequire a ligand to be attached to the AFM tip, preferably by a distensible poly(ethylene glycol) (PEG) chain between themeasuring tip and the ligand molecule. The PEG chain greatly facilitates specific binding of the ligand to immobile receptorsites on the sample surface.

The present study contributes to tip-PEG-ligand tethering in three ways: (i) a convenient synthetic route was found to prepareNH2–PEG–COOH which is the key intermediate for long heterobifunctional crosslinkers; (ii) a variety of heterobifunctionalPEG derivatives for tip-PEG-ligand linking were prepared from NH2–PEG–COOH; (iii) in particular, a new PEG crosslinkerwith one thiol-reactive end and one terminal nitrilotriacetic acid (NTA) group was synthesized and successfully used to tether

Abbreviations: AFM, atomic force microscope (or microscopy); Boc,O-tert-butyloxycarbonyl; Boc2O, O,O′-di-tert-butyldicarbonate;biotin–PEG–NHS, seeFig. 2; BSA, bovine serum albumin; DCC,N,N′-dicyclohexylcarbodiimide; DIEA,N,N-diisopropyl-N-ethylamine;DMF, N,N-dimethylformamide; DTT, 1,4-dithiothreitol; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; ESI-MS, electrospray mass spectrum (or spectrometry); glu, glutaryl residue; HRV2, human rhinovirus serotype 2; LC-SMCC,O-(N-succinimidyl)-6-{[4-(N-maleimidomethyl)-cyclohexane-1-carbonyl]-amino}-hexanoate; lys-DA, lysine-N�,N�-diacetic acid:N-(5-amino-1-carboxypentyl)iminodiacetic acid; mal–PEG–NHS, seeFig. 2; MBP-VLDLR1-3, fusion product of maltose-binding protein with thethree N-terminal repeats of the very-low density lipoprotein receptor; NH2–PEG–NH2, O,O′-bis(2-aminopropyl)poly(ethylene glycol) 800;NH2–PEG–COOH,N-glutaryl derivative of NH2–PEG–NH2; NHS,N-hydroxysuccinimide; NMR, nuclear magnetic resonance; NTA, nitrilotri-acetate; PDP, 3-(2-pyridyl)-dithiopropionyl group; PDP-OH, 3-(2-pyridyl)-dithiopropionic acid; PDP–PEG–NHS, seeFig. 2; PDP–PEG–NTA,seeFig. 2; PEG, poly(ethylene glycol); RT, room temperature; SATP,O-succinimidyl S-acetylthiopropionate; SPDP,O-succinimidyl 3-(2-pyridyl)-dithiopropionate; SMRFM, single molecule recognition force microscope (or microscopy); TEA,N,N,N-triethylamine; Tris, tris-(hydroxymethyl)-aminomethane; VLDLR, very-low density lipoprotein receptor; VS–PEG–NHS, seeFig. 2; X–PEG–NH2, impurity ofNH2–PEG–NH2 with one inert terminus; X–PEG–X, PEG with two inert end groups

∗ Corresponding author. Tel.:+43-732-2468-9271; fax:+43-732-2468-9280.E-mail address: [email protected] (H.J. Gruber).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2003.08.041

102 C.K. Riener et al. / Analytica Chimica Acta 497 (2003) 101–114

His6-tagged protein molecules to AFM tips via noncovalent NTA–Ni2+–His6 bridges. The new crosslinker was applied to linka recombinant His6-tagged fragment of the very-low density lipoprotein receptor to the AFM tip whereupon specific dockingto the capsid of human rhinovirus particles was observed by force microscopy. In a parallel study, the specific interaction of thesmall GTPase Ran with the nuclear import receptor importin�1 was studied in detail by SMRFM, using the new crosslinkerto link His6-tagged Ran to the measuring tip [Nat. Struct. Biol. (2003), 10, 553–557].© 2003 Elsevier B.V. All rights reserved.

Keywords: Atomic force microscope; Recognition force spectroscopy; Human rhinovirus; Poly(ethylene glycol); Heterobifunctionalcrosslinker

1. Introduction

Specific recognition and binding between biomole-cules is a key element of biological systems. A va-riety of techniques have been developed to studybiospecific interactions on various scales of size andtime (see[1] for a review). Very detailed results areobtained by single molecule recognition force mi-croscopy (SMRFM) which can measure interactionforces on the single molecule level. The basic toolfor SMRFM is a measuring tip whose apex has beenfunctionalized with one or few probe molecules thatcan recognize a specific type of target molecules. Ina number of laboratories, AFM tips have been coatedwith probe molecules which were either stronglyadsorbed[2,3] or covalently bound[4–9].

As exemplified by a number of studies[8,10–19],the insertion of a 6–8 nm long PEG spacer betweenthe probe molecule and the apex of the measuringtip affords significant technical improvements: (i) thePEG spacer allows the probe molecule to freely reori-ent for unconstrained receptor–ligand recognition; (ii)the probe molecule can “scan” a large surface for tar-get molecules during tip-substrate encounter; (iii) theprobe molecule escapes the danger of being crushedbetween tip and substrate during hard contact; (iv)most importantly, the soft and nonlinear elasticity ofthe PEG tether allows to clearly distinguish betweenunspecific and receptor-specific binding of the tip. Thenonlinear elasticity of the 6–8 nm long PEG spacerupon stretching up to the moment of receptor–ligandbond rupture has thoroughly been characterized ina preceding AFM study[16] and quantitatively ana-lyzed according to theoretical models[20–22].

The standard procedure for tip-PEG-probe linkingis depicted inFig. 1. In the first step, the amino-functionalized AFM tip [8,23] is reacted with theNHS-ester terminus of the PEG crosslinker. Usually

about 1–3 PEG tentacles are attached which can reachbelow the apex of the AFM tip[13,23]. In the secondstep, the pyridyldithio group at the outer end of PEGreacts with a free thiol group on the probe molecule,yielding a stable disulfide, while 2-mercaptopyridineis released which immediately tautomerizes into2-thiopyridone[24,25].

The original synthesis[24] of the long crosslinkerin Fig. 1 had several drawbacks. It required largeamounts of the expensive reagent SPDP in the first

Fig. 1. Standard procedure for the tethering of single antibodymolecules to AFM tips. Amino groups are generated on the tipby one of three characterized procedures[23]. In step 1, theseamino groups react with the NHS ester functions of the PEGcrosslinker molecules, yielding stable amide bonds. In step 2, thepyridyldithio group on the outer end of PEG reacts with a freethiol group on the probe molecule, resulting in a stable disulfidebond while 2-mercaptopyridine is released.

C.K. Riener et al. / Analytica Chimica Acta 497 (2003) 101–114 103

step, yields were moderate, and the synthetic routewas not adaptable to the synthesis of similar AFMcrosslinkers with other coupling functions. Theseproblems were overcome by a new synthetic routein which the central element (NH2–PEG–COOH, seeFig. 2) was synthesized first[23]. As can be seen fromFig. 2, NH2–PEG–COOH is the key intermediate for

Fig. 2. NH2–PEG–COOH as a precursor to various long heter-obifunctional crosslinkers. Biotinylation of NH2–PEG–COOH (a)and activation of the terminal carboxyl (b) gave biotin–PEG–NHS[23]. In the reaction steps c, d, or e, three different thiol-reactivefunctions were attached to the NH2-group of NH2–PEG–COOH,followed by activation of the terminal carboxyl groups as NHSester (b). In case of PDP–PEG–NHS, the crosslinker was furtherreacted with lys-DA to introduce the NTA function (f), useful forthe linking of His6-tagged probe molecules (seeFig. 4).

Fig. 3. Synthesis of the key intermediate (NH2–PEG–COOH) forheterobifunctional PEG derivatives. (a) 0.2 equivalents of glutaricanhydride; (b) excess of Boc2O; (c) excess of methylamine; (d)chromatography on QAE Sephadex A-25; (e) 98% formic acid;(f) rechromatography on QAE Sephadex A-25.

a variety of long flexible crosslinkers. Nevertheless,we were not fully satisfied with the method of syn-thesizing NH2–PEG–COOH because in the first andthird chromatographic step it was tricky to achievehigh purity without sacrificing too much product.

In the present study, NH2–PEG–COOH has beensynthesized by a new method (seeFig. 3) which re-quires only two chromatographic steps in water andgives pure product in high yield. Furthermore, a vari-ety of crosslinkers for tip-PEG-probe conjugation havebeen synthesized, including a new PEG derivative withan NTA group for noncovalent linking of His6-taggedproteins (seeFig. 2). The use of the latter is exempli-fied by tethering of a His6-tagged ectodomain frag-ment of the VLDLR to an AFM tip and measurementof specific adhesion events with mica-bound humanrhinovirus particles seeFigs. 4 and 5.

2. Materials and methods

2.1. Materials

Analytical grade materials were used if theywere commercially available. The starting mate-


rials were of the highest available purity. Unlessstated otherwise, buffer components and organicsolvents, as well as acetic acid, ammonia solution(32% in water), DCC, D2O, formic acid, hydroxy-lamine hydrochloride, LiChroprep RP-18 gel, methy-lamine (40% in water), trifluoroacetic acid, wereobtained from Merck (Germany). DIEA was pur-chased from Aldrich (Austria). Sephadex G-25M andQAE-Sephadex A-25 were obtained from AmershamBiosciences (UK). Chloroform and toluene were pur-chased from J.T. Baker (The Netherlands). Divinylsul-fone, O,O′-bis(2-aminopropyl)poly(ethylene glycol)800, ninhydrin, and TEA were obtained from Fluka(Germany). Muscovite mica sheets were suppliedby Christiane Gröpl (Austria). LC-SMCC was ob-tained from Molecular Biosciences (Colorado). Si3N4measuring tips were bought from Park Scientific (Sun-nyvale, CA). Boc2O, CDCl3, 2,2′-DTDP, ethanolam-monium hydrochloride, glutaric anhydride, NHS,and Tris base were obtained from Sigma (Austria).Lys-DA was bought from Toronto Research Chem-icals (Canada). SPDP was synthesized as described[26].

2.2. Buffers

Buffer A contained 100 mM NaCl, 50 mMNaH2PO4, and 1 mM EDTA (pH 7.5 adjusted withNaOH). Buffer B contained 150 mM NaCl, 0.1 mMNiCl2, 1 mM CaCl2, and 50 mM Tris base (pH 7.6adjusted with HCl). Buffer C contained 150 mMNaCl and 100 mM EGTA (pH 7.6 adjusted with Trisbase). Buffer D contained 50 mM Tris base and 5 mMNiCl2 (pH 7.6 adjusted with HCl). PBS contained150 mM NaCl and 5 mM NaH2PO4 (pH 7.4 ad-justed with NaOH). Hydroxylamine reagent (500 mMhydroxylamine·HCl, 25 mM EDTA, pH 7.5) wasprepared as described[24].

2.3. Thin layer chromatography

Merck plastic sheets (silica gel 60) without fluores-cent indicator were used. Eluents I, II, and III con-tained 70 parts of chloroform, 30 parts of methanol,and 4 parts of concentrated ammonia, or water, oracetic acid, respectively. Amino groups were specif-ically stained with ninhydrin (Boc–NH-groups werealso stained after∼1 min at∼100◦C, due to thermal

deprotection), all other components were visualized iniodine vapor.

2.4. Synthesis of NH2–PEG–COOH

In a 250-ml round bottomed flask,O,O′-bis(2-aminopropyl)poly(ethylene glycol) 800 (9.1 g,∼9.6mmol) was dissolved in chloroform (125 ml) andstirred under an argon atmosphere. The solution wascooled in liquid nitrogen until the solution beganto freeze (−35◦C solution temperature). The liquidnitrogen bath was removed and a solution of glu-taric anhydride (225 mg, 1.97 mmol) in chloroform(10 ml) was slowly added with stirring under argonatmosphere. The temperature rose to+10◦C withinthe first hour and to+20◦C within the second hour.After 3 h, the solvent was evaporated on the rotavapat 30◦C bath temperature. The honey-like productwas dissolved in methanol (75 ml) and stirred ina 3-necked 250-ml round-bottomed flask under ar-gon atmosphere. An argon-bubbled solution of TEA(2.25 ml, 16 mmol) in methanol (3.5 ml) was addedwith stirring. Subsequently, an argon-bubbled solutionof Boc2O (4.36 g, 20 mmol) in methanol (7.5 ml) wasadded dropwise. The completion of the reaction waschecked by TLC in chloroform/methanol/acetic acid(80/20/2, v/v/v). The excess of Boc2O was quenchedby dropwise addition of a mixture of methylamine(1.73 ml of a 40% solution, 20 mmol) and methanol(7.5 ml). After 15 min of stirring, the solution wascooled to 0◦C and the solvent was removed by ro-tary evaporation. The residue was dried at 1–10 Pafor 3 h, dissolved in water (110 ml) and the pH wasadjusted to 7.5 with dilute NaOH. The solution wasfrozen to the walls of a 2-l flask and lyophilized toremove excess of methylamine. The dry residue wasdissolved in water (200 ml final volume), the pH wasreadjusted to 7.5, and the solution was loaded at aflow of 4 ml min−1 on an anion exchange column(QAE Sephadex A-25, 5 cm diameter, 17.5 cm bedheight, chloride form, stored in 20% ethanol, precon-ditioned with >2 bed volumes of distilled water at aflow of 1 ml min−1) while collecting 95 ml fractions(# 1–2). The column was washed with water (384 ml,fractions # 3–6) until all uncharged PEG derivativeswere eluted, as detected by spotting of eluate on TLCplates and developing in eluent I. Elution was contin-ued with 20 mM Tris (adjusted to pH 7.5 with HCl,


1460 ml, fractions # 8–22). The column was regen-erated from dicarboxylic acids by washing with 5 MNaCl, 50 mM Tris (pH 7.5 adjusted with HCl, 700 ml)and re-equilibrated in distilled water (1000 ml) forthe second column step. Fractions # 11–18 werepooled and taken to dryness by rotary evaporationwithout heating, using a strong vacuum source. Thedry residue was dissolved in 50 ml of 6 M NaCl andextracted with chloroform (1× 50 ml, 2× 25 ml). Thecombined organic phases were dried with Na2SO4,filtered, and taken to dryness. The residue was driedat 1–10 Pa for 3 h, yielding 1.38 g of a honey-likeproduct which was split into two portions. The1HNMR spectrum showed the expected singlet for theBoc group at 1.43 ppm in addition to the other signalsof NH2–PEG–COOH.

For cleavage of the Boc-group, one portion(741 mg) of the above product was dissolved in 2.6 mlof water and 17.3 ml of formic acid was added. After3 h of stirring at RT, 17.3 ml of toluene was addedand the mixture was evaporated to dryness. Evapora-tion was repeated twice (with 10 and 5 ml of toluene)and the product was dried at 1–10 Pa overnight. Thehoney-like residue (619 mg) was dissolved in water(20 ml) and the pH was adjusted to 7.5 with NaOH.The solution was loaded on the regenerated andre-equilibrated anion exchange column (see above).The column was eluted with water (600 ml) whilecollecting 20-ml size fractions. Subsequently, the col-umn was regenerated with 5 M NaCl, 50 mM Tris(pH 7.5 adjusted with HCl, 700 ml), washed withwater (1000 ml) and either reused or stored in 20%ethanol. Fractions # 8–15 were pooled and taken todryness by rotary evaporation without heating, us-ing a strong vacuum source. The dry residue wasdissolved in 20 ml 6 M NaCl and extracted with chlo-roform (4× 15 ml). The combined organic phaseswere dried with Na2SO4, filtered, taken to dryness,and the residue was dried at 1–10 Pa for 3 h, yielding0.534 g (∼0.50 mmol) of a honey-like product whichwas pure by TLC (RI

f = 0.27–0.53,RIIf = 0.23–0.48,

RIIIf = 0.26–0.51).1H NMR (200 MHz, CDCl3) δ: 1.06–1.2 (∼10H,

m, NH2–CH<(CH3)–CH2–O and NH2–CH<(CH 2–CH 3)–CH2–O of PEG), 1.94 (2H, quin,J = 7 Hz,CO–CH2–CH 2–CH2–CO, position �′ in glu),2.26 (2H, t, CH2–CH 2–COOH), 2.36 (2H, t,J = 7 Hz, CH2–CH 2–CO–NH), 3.35–3.54 (6H, m,

N–CH<(CH3)–CH2–O and N–CH<(CH2–CH3)–CH 2–O of PEG), 3.54–3.70 (72H, s, O–CH 2–CH 2–O,∼18 ethylene glycol units), 4.1 (1H, broad s,CH2–CO–NH–CH<(C)–CH2–O), 7.26 (s, CDCl3),7.88 (3H, broad s, NH3+). In the preceding study ithas been shown that∼50% of the termini in com-mercial NH2–PEG–NH2 were 2-aminobutyl and not2-aminopropyl groups and that the PEG chain con-sisted of 18± 3 ethylene glycol units[23].

2.5. Synthesis of mal–PEG–NHS

A solution of NH2–PEG–COOH (26.7 mg,∼25�mol) in DMF (1.1 ml) was stirred under an ar-gon atmosphere and LC-SMCC (14 mg, 37.5�mol)was added. The acylation reaction was started by ad-dition of DIEA (14.5�l, 75�mol) and stirring underargon was continued. For monitoring the reactionby TLC, a small aliquot was evaporated at 1–10 Paand redissolved in a drop of chloroform beforespotting. After 30 min, traces of ninhydrin-reactiveNH2–PEG–COOH were still visible while after60 min the turnover of educt was complete. The mix-ture was frozen in liquid nitrogen and upon thawing,the solvent was removed with stirring at 1–10 Pa.

The dry residue was purified by low-pressure re-versed phase chromatography on Merck LiChroprepRP-18 gel (bed size 1.5 cm× 3 cm, flow 2 ml min−1)[27]. Solvent A consisted of water containing 0.1%acetic acid and solvent B was a 5/2 mixture (v/v)of ethanol and 1-propanol[28]. The crude productwas loaded in 3 ml of 5% solvent B and the col-umn was eluted with 10 ml portions each of 5, 10,15, 20, 25, 35, and 50% solvent B while collect-ing 10-ml size fractions at the same time. The col-umn was regenerated with 80% solvent B containing0.1% TFA and stored in 20% ethanol. The pure in-termediate (mal–PEG–COOH) was found in fractions# 5–6 which were combined, taken to dryness anddried at 1–10 Pa overnight, yielding 21 mg (∼15�molmal–PEG–COOH) of viscous product which was pureby TLC (RI

f 0.36–0.57,RIIf 0.56–0.70,RIII

f 0.56–0.67).1H NMR: (500 MHz, in CDCl3, 1-dim 1H

and 2-dim 1H–1H– and 13C–1H–COSY; [29]) δ

(ppm): 0.99 (2H, dq, 13/3 Hz, axial ring positions3 and 5 [27]), 1.08–1.18 (∼10 H, m, NH2–CH<

(CH 3)–CH2–O and NH2–CH<(CH 2–CH 3)–CH2–Oof PEG), 1.33 (2H, quin, 7.5 Hz, C–CH 2–C position


� in aminohexanoyl), 1.42 (2H, dq, 13/3 Hz, axial ringpositions 2 and 6), 1.50 (2H, quin, 7.5 Hz, C–CH 2–Cposition� in aminohexanoyl), 1.64 (2H, quin, 7.5 Hz,C–CH 2–C position� in aminohexanoyl), 1.66–1.69(1H, m, axial ring position 4), 1.73 (2H, dd, 13/3 Hz,equatorial ring positions 3 and 5), 1.87 (2H, dd,13/3 Hz, equatorial ring positions 2 and 6), 1.96 (2H,quin, 7.5 Hz, CH2–CH 2–CH2, position�′ in glu), 2.00(1H, tt, 13 Hz, 3 Hz, axial ring position 1), 2.18 (2H, dt,7.5 Hz, C–CH 2–CO–N position� in aminohexanoyl),2.26–2.31 (2H, m, NH–CO–CH 2–CH2–CH2 position�′ in glu), 2.36–2.41 (2H, m, CH2–CH2–CH2–COOHposition �′ in glu), 3.19–3.26 (2H, m, 7.5 Hz,O–CO–CH 2–CH2 position ε in aminohexanoyl),3.36 (2H, d, 7 Hz, >N–CH 2–C< bridge betweenmaleimide and the ring), 3.40–3.58 (6H, m, N–CH<

(CH3)–CH 2–O and N–CH<(CH2–CH3)–CH 2–Oof PEG), 3.60–3.70 (72H, s, O–CH 2–CH 2–O,∼18 ethylene glycol units[23]), 4.09–4.14 (2H, m,CH2–CO–NH–CH2 of both PEG termini), 6.69 (2H,s, N–CH=CH–N of mal), 7.26 (s, CDCl3). Due to thelongT1 relaxation time of the olefinic hydrogen atoms,a 25 s delay time between pulses was required toobtain a correct integral for the maleimide group[30].

13C NMR: (500 MHz, in CDCl3, 1-dim 13C and2-dim 13C–1H–COSY; [29,31] δ (ppm): 17.6 (16.6)(N–CH<(CH2)–CH3 of PEG-2(3)-amino-butylterm)21.0 (C–CH2–C position�′ in glu), 25.0 (C–CH2–Cposition � in LC), 26.2 (C–CH2–C position � inLC), 28.8 (positions 2 and 6 in cyclohexane), 29.0(C–CH2–C position� in LC), 29.9 (positions 3 and5 in cyclohexane), 32.9 (C–CH2–CO–O position�′in glu), 35.2 (N–CO–CH2–C position�′ in glu), 36.1(C–CH2–CO–N position� in LC), 36.4 (position 4 incyclohexane), 39.0 (O–CO–CH2–C positionε in LC),43.7 (>N–CH2–C< bridge between maleimide andcyclohexane), 45.2 (position 1 in cyh), 45.3 (N–CH<

of PEG-2(3)-aminobutylterm) 70.5 (O–CH2–CH2–O18 units of PEG), 75.0 (73.9) (N–CH–CH2–O ofPEG-2(3)-aminobutylterm), 77.0 (CDCl3 t, J =32 Hz), 134.0 (N–CH=CH–N of mal), 171.0 (>C=O).

The above free carboxylic acid form (mal–PEG–COOH, 8.4 mg, 6�mol) was dissolved in ethyl ac-etate (160�l) and 1.4 mg of NHS (12�mol) wasdissolved in 120�l of ethyl acetate and 90�l thereofwere added to mal–PEG–COOH. Then, 2.5 mg ofDCC was also dissolved in 120�l of ethyl acetate,and 90�l thereof were pipetted into the reactions

mixture. After stirring at RT overnight, the solutionwas filtered from dicyclohexylurea and the filtrate wasevaporated, yielding 7.9 mg of viscous product whichwas pure by TLC (RI

f 0.69–0.85,RIIf 0.65–0.71,RIII

f0.71–0.81), except for contamination with free NHS(RI

f 0.02–0.13). The product gave the same NMRspectrum as the free carboxylic acid form, except forthe additional singlet at 2.86 ppm (NHS residue) andthe typical shift of the�-CH2 group next to the ter-minal COOH group from 2.38 ppm (free acid form)to 2.50 ppm (NHS ester form).

2.6. Synthesis of VS–PEG–NHS

NH2–PEG–COOH (49 mg,∼45�mol) was dis-solved in 1.5 ml of chloroform and stirred under anargon atmosphere and TEA (21�l, 150�mol) wasadded. SPDP (21 mg, 65�mol) was dissolved inchloroform (1 ml) and added to the mixture undercontinued stirring under argon. After 10 min, TLCshowed complete conversion of NH2–PEG–COOHinto PDP–PEG–COOH and the solvent was removed.The residue was thoroughly suspended in buffer A(2 ml). Undissolved SPDP was removed by cen-trifugation (10,000 g, 10 min) and by filtering thesupernatant (Millex GS, 0.2�m) into a 10 ml measur-ing cylinder where it was bubbled with argon. DTT(14 mg, 90�mol) was added and bubbling with argonwas continued for 10 min. After acidification to pH4.5 with acetic acid, 22.5�l (225�mol) of divinylsul-fone (harmful!) was pipetted into the solution and thepH was gradually raised to 7 with 10% Na2CO3 withcontinued argon bubbling. After 2 h the reaction wasstopped by acidification to pH 4.5 with 1 M HCl. Themixture was diluted to 4 ml with water and loaded ona Sephadex G-25F column (2.5 cm × 44 cm, condi-tioned in distilled water). The column was eluted withdistilled water at a flow of 2.7 ml min−1 while collect-ing 8-ml size fractions. The column was regeneratedwith 160 ml of 3 M NaCl and either washed with wa-ter for another use or stored in a solution containing40 mM Tris base and 5 mM sodium azide. Fractionscontaining the product (# 12–19) were pooled andsaturated with NaCl. The product was extracted intochloroform (4× 30 ml), the combined organic phaseswere dried over Na2SO4, filtered, and the filtrate wasevaporated. Overnight drying at 1–10 Pa gave 30 mgof viscous product (∼24�mol VS–PEG–COOH)


which was pure by TLC (RIf 0.24–0.45,RII

f 0.52–0.72,RIII

f 0.53–0.69). It was dissolved in dry ethyl acetate(0.4 ml), NHS (52.6 mg, 24�mol) was added andcompletely dissolved with stirring. DCC (29.3 mg,24�mol) was added and the solution was stirred atRT overnight. Dicyclohexylurea was filtered off andthe filtrate was evaporated, yielding 25 mg of viscousproduct (∼18�mol VS–PEG–NHS) which was pureby TLC (RI

f 0.55–0.68,RIIf 0.56–0.65,RIII

f 0.56–0.77).1H NMR: (500 MHz,1H-spectrum, 2-dim1H–1H–

COSY in CDCl3) � (ppm): 1.03–1.24 (∼10 H, m,NH2–CH<(CH 3)–CH2–O and NH2–CH<(CH 2–CH 3)–CH2–O of PEG), 2.11 (2H, quin,J = 7 Hz,CH2–CH 2–CH2, position �′ in glu), 2.28 (2H, t,J = 7 Hz, NH–CO–CH 2–CH2, position �′ in glu),2.45 (2H, t, J = 7 Hz, NH–CO–CH 2–CH2–S),2.69 (2H, t,J = 7 Hz, CH2–CH 2–CO–NHS), 2.78–2.97 (4H, m, NH–CO–CH2–CH 2–S and S–CH 2–CH2–SO2), 2.85 (4H, s, NHS residue), 3.21–3.33(2H, m, S–CH2–CH 2–SO2) 3.34–3.56 (6H, m,N–CH<(CH3)–CH 2–O and N–CH<(CH2–CH3)–CH 2–O of PEG), 3.64 (72H, s, O–CH2–CH2–O,∼18 ethylene glycol units[23]), 4.03–4.19 (2H, m,CH2–CO–NH–CH2 of both PEG termini), 6.21 (1H,d1, 3Jcis = 9.8 Hz, SO2–CH=CH cisH), 6.45 (1H,d1, 3Jtrans = 16.6 Hz, SO2–CH=CHH trans), 6.70(1H, dd1, 3Jtrans = 16.6 Hz and 3Jcis = 9.8 Hz,SO2–CH=CH2), 7.26 (s, CDCl3).

2.7. Synthesis of PDP–PEG–NHS

NH2–PEG–COOH (474 mg,∼0.45 mmol) wasdissolved in chloroform (7 ml) and stirred underargon atmosphere. TEA (0.21 ml, 1.5 mmol) andSPDP (205 mg, 0.66 mmol) were added. The reac-tion was monitored by TLC to make sure that allninhydrin-reactive educt was derivatized with SPDP.Then, the solvent was evaporated, the residue wasdried at 1–10 Pa for 3 h and thoroughly suspendedin buffer A (12.5 ml). After centrifugation (20 minat 10,000 g) the supernatant was filtered (Millex GS,0.2�m) and chromatographed on a gel filtration col-umn (Sephadex G-25M, 2.5 cm× 44 cm) in distilledwater at a flow of 2.7 ml min−1 while collecting 8 mlfractions. The column was regenerated with 160 ml of3 M NaCl and either re-equilibrated in water for an-other use or stored in a solution containing 40 mM Trisbase and 5 mM sodium azide. Elution of the product

was detected by reductive cleavage of the PDP groupwith 2-mercaptoethanol and measurement of UV ab-sorption at 343 nm (ε = 8080 M−1 cm−1 [32]). In par-ticular, 50�l of column eluate was mixed with 1140�lbuffer A, 25�l of 0.5 M 2-mercaptoethanol was added,andA343 was measured after∼5 min. Fractions con-taining the product (# 12–17) were pooled and takento dryness by rotary evaporation without heating, us-ing a strong vacuum source. The residue was dried at1–10 Pa overnight, yielding 412 mg of viscous productwhich was pure by TLC (RI

f 0.33–0.53,RIIf 0.65–0.81,

RIIIf 0.67–0.83). 315 mg of this intermediate was dis-

solved in ethyl acetate (3 ml), 52.6 ml of NHS wasadded and completely dissolved with stirring. Then,29.3 mg of DCC was added and the solution wasstirred at RT overnight. Dicyclohexylurea was filteredoff, the filtrate was evaporated, and dried at high vac-uum (1–10 Pa) yielding 319 mg (∼0.24 mmol) of vis-cous product which was pure by TLC (RI

f 0.40–0.55,RII

f 0.69–0.88,RIIIf 0.64–0.80). The molecular struc-

ture was confirmed by ESI-MS (data not shown, com-pare reference Riener 2003). Moreover, no indicationof a dimeric crosslinker (NHS–PEG–S–S–PEG–NHS)was observed in MALDI-TOF-MS (data not shown).

1H NMR: (200 MHz, in CDCl3) δ (ppm): 1.04–1.26(∼10 H, m, NH2–CH<(CH 3)–CH2–O and NH2–CH<

(CH 2–CH 3)–CH2–O of PEG), 1.96 (2H, quin,7.2 Hz, CH2–CH 2–CH2, position�′ in glu), 2.23–2.48(4H, m(2t), NH–CO–CH 2–CH2–CH 2–CO–NH po-sition �′ and�′ in glu), 2.56 (2H, t, 6.4 Hz, NH–CO–CH 2–CH2–S–S, position 2 in prop), 2.85 (4H, s,CO–CH 2–CH 2–CO of NHS-ester), 3.06 (2H, t,6.5 Hz, NH–CO–CH2–CH 2–S–S, position 3 in prop),3.34–3.56 (6H, m, N–CH<(CH3)–CH 2–O andN–CH<(CH2–CH3)–CH 2–O of PEG), 3.66 (72H, s,O–CH 2–CH 2–O,∼18 units of PEG), 3.95–4.15 (2H,m, CH2–CO–NH–CH2 of both PEG termini), 7.12(1H, m(dd), position H5 of pyridyl residue), 7.26 (s,CDCl3), 7.65 (2H, m(dt), position H-3,4 of pyridylresidue), 8.45 (1H, m(dd), position H6 of pyridylresidue).

2.8. Synthesis of PDP–PEG–NTA

A suspension of lys-DA (15 mg, 50�mol) inmethanol (0.2 ml) was stirred under argon. Uponaddition of TEA (42�l, 300�mol) the solid wasdissolved. This solution was slowly dropped into a


solution of PDP–PEG–NHS (109 mg, 80�mol) inmethanol (0.75 ml) while stirring under argon atmo-sphere (∼1 drop min−1). The dropping funnel wasrinsed with 0.5 ml of methanol which were added tothe reactions mixture. After another 10 min of stir-ring the solvent was removed by rotary evaporation.The residue was dried at 1–10 Pa for 2 h, redissolvedin 10 ml of 1 mM NaH2PO4 (pre-adjusted to pH 7with NaOH) and centrifuged for 20 min at 2500× g).The supernatant was filtered (Millex GS, 0.2�m) andloaded on an anion exchange column (QAE SephadexA-25, 1.5 cm×12.5 cm, chloride form, conditioned indistilled water) at a flow of 1 ml min−1. After washingwith 2 bed volumes of distilled water, the column waseluted with a multiple step gradient of HCl in water.The column was successively washed with 16 ml por-tions of 10, 20, 40, 80, 160, 320, and 640 mM HClwhile collecting 4-ml size fractions. The column wasregenerated with 160 ml of 3 M NaCl, reconditionedwith 3 bed volumes of water and either reused orstored in 20% ethanol. Elution of the product wasdetected by reductive cleavage and measurement ofreleased 2-thiopyridone by UV absorption at 343 nm,as described above (seeSection 2.7). Fractions #18–20 were pooled, and purified on a reversed phasecolumn (Merck LiChroprep RP-18, 1.5 cm×5 cm), asdescribed above for mal–PEG–COOH. Fractions con-taining the product were detected by UV absorptionmeasurement (see above), pooled, and taken to dry-ness by rotary evaporation. The residue was dried at1–10 Pa overnight, yielding viscous product (53.5 mg,∼36�mol) which was pure by TLC (RI

f 0.00–0.12,RII

f 0.00–0.20,RIIIf 0.00–0.12).

1H NMR: (500 MHz,1H-spectrum, 2-dim1H–1H–COSY in D2O) δ (ppm): 1.08–1.19 (∼10H, m, NH2–CH<(CH 3)–CH2–O and NH2–CH<(CH 2–CH 3)–CH2–O of PEG), 1.46–1.63 (2H, m, CH2–CH 2–CH2–CH(CO)–N< position�I , �II in lys-DA residue) 1.60(2H, CO–NH–CH2–CH 2–CH2, position� in lys-DAresidue), 1.89 (2H, quin, 7.2 Hz, CH2–CH 2–CH2,position �′ in glu), 1.85–2.03 (2H, m(qd), CH2–CH 2–CH(CO)–N< position �I , �II in lys-DAresidue), 2.23–2.31 (4H, m(2t), NH–CO–CH 2–CH2–CH 2–CO–NH position �′ and �′ in glu), 2.71(2H, t, 6.4 Hz, NH–CO–CH 2–CH2–S–S), 3.16(2H, t, 6.4Hz, NH–CO–CH2–CH 2–S–S), 3.22 (2H,m(t), CO–NH–CH 2–CH2, position ε in lys-DA),3.38–3.59 (6H, m, N–CH<(CH3)–CH 2–O and

N–CH<(CH2–CH3)–CH 2–O of PEG), 3.65 (72H,s, O–CH 2–CH 2–O, ∼18 ethylene glycol units[23]), 3.88–4.01 (5H, m, >N–CH 2–COOH (2×) andCH2–CH (COOH)–N< of lys-DA residue), 3.95–4.15(2H, m, CH2–CO–NH–CH2 of both PEG termini),4.80 (bs,HOD), 7.63 (1H, t(dt)1, position H5 ofpyridyl residue), 8.11 (1H, d(dd)1, position H3 ofpyridyl residue), 8.21 (1H, t(dt)1, position H4 ofpyridyl residue), 8.59 (1H, d(dd)1, position H6 ofpyridyl residue).

2.9. Imaging of HRV2 particles on mica

HRV2 viral particles were prepared and purified asdescribed[33]. Freshly cleaved mica was incubatedwith 100�l of ∼0.1 mg ml−1 HRV2 in buffer D for15 min. After 3 washing steps with the same buffer,the sample was imaged with a magnetically driven dy-namic force microscope (Molecular Imaging, Phoenix,AZ) in the same buffer at room temperature. The can-tilever (Maclevers, Molecular Imaging) had a springconstant of 0.1 N m−1. The peak-to-peak oscillationamplitude was 5 nm at 7 kHz driving frequency. Thefeedback was adjusted to 30% amplitude reduction.

2.10. Measurement of single unbinding eventsbetween VLDLR1-3 and HRV2

His6-tagged VLDLR1-3 was coupled to AFM tips(Park Scientific, Sunnyvale, CA), using a multi-stepprotocol. First, the tips were functionalized withethanolamine·HCl at RT [23]. Second, these aminogroups were derivatized with SATP (0.5%, w/v) andTEA (0.5%, v/v) in chloroform solution (1.5 h atRT). After washing with chloroform (3×) and dry-ing with nitrogen gas, the tip was immersed in anaqueous solution prepared from 9 volumes of bufferA containing 3 mg ml−1 of PDP–PEG–NTA and 1volume of hydroxylamine reagent which had beendeoxygenated with argon gas. After 1 h of incuba-tion at room temperature, the NTA functions on thetips were loaded with Ni2+ by 20 min incubationin buffer D. The cantilevers were washed in PBS(3×) and incubated in PBS solution containing 1�MHis6-tagged MBP-VLDLR1-3 (a fusion product be-tween maltose-binding protein and the first threeligand binding repeats of the human very-low density


lipoprotein receptor, prepared as described in[34])for 40 min, followed by three washing cycles in PBS.

Force-displacement measurements were performedwith a Macmode PicoSPM Microscope (Molecu-lar Imaging) equipped with a commercial fluid celland with a Molecular Imaging Macmode interfacedriving a Nanoscope IIIa controller (Digital Instru-ments, Santa Barbara, CA). Force–distance cycleswith receptor-modified tips and virus-loaded micawere carried out in buffer B. Ca2+ ions are requiredto maintain the ligand binding repeats in their nativeconformation. Therefore, in control experiments, theinteraction between virus and MBP-VLDLR1-3[35]was specifically eliminated by complexation withEGTA (30 min incubation in buffer C). Force–distancecycles were performed with az-range of 100 nm and afrequency of 0.3 Hz. The spring constants of the can-tilevers were determined by the thermal noise method[36,37]. The force–distance cycles were analyzed asdescribed in detail[38].

3. Results and discussion

3.1. Preparation of pure NH2–PEG–COOH

From Fig. 2 it can be seen that NH2–PEG–COOHis a key intermediate for the syntheses of many longheterobifunctional crosslinkers with a PEG spacer ele-ment. In the present study, the synthetic route depictedin Fig. 3 was found to yield NH2–PEG–COOH withthe desired purity, i.e. each PEG molecule had exactlyone NH2-group and one COOH, as judged from TLC,ESI-MS, and1H NMR.

The commercial educt wasO,O′-bis(2-aminopro-pyl)poly(ethylene glycol) 800 which is known to havean average number of 18 ethylene glycol units andonly about 1.6 NH2-groups per PEG chain[8,23,39],in other words,≤20% of the PEG diamine was presentas X–PEG–NH2, and≤4% as X–PEG–X, “X” denot-ing some unknown terminal group which was found tobe uncharged between pH 2 and 12[23]. No econom-ical way was found to eliminate the PEG moleculeswith one or two “missing NH2-groups”[23] but fortu-nately the glutarylated derivatives of these impuritieswere easily removed by ion exchange chromatographyon QAE Sephadex A-25 in aqueous solution. This gelhas an exceptionally high charge density (0.5 mol l−1

bed volume, as compared to 0.1–0.2 mol in mostother gels) and tightly binds monocarboxylate deriva-tives of PEG 800 from salt-free solution. While QAESephadex A-50 was found to bind a higher amount ofnegatively charged PEG per gram of dry gel, the A-50gel was inferior to A-25 in practice because the bedvolume of A-50 expanded by an order of magnitudein distilled water (the binding condition) as comparedto 10−2 to 10−1 M salt (the desorption condition).This led to poor effective capacity of Sephadex A-50column beds and to great technical difficulties (datanot shown).

One critical observation for devising the procedurein Fig. 3 was the following. The binding capacity ofQAE Sephadex A-25 for PEG 800 monocarboxylateswas greatly diminished when a significant fraction ofdicarboxylic acid salt was present. For this reason,the cheap starting material NH2–PEG–NH2 was sta-tistically derivatized with as little as 0.2 equivalentsof glutaric anhydride per 2 equivalents of acidimetri-cally determined NH2-groups (see[23] for the acidi-metric titration) which provided for a low fractionof HOOC–PEG–COOH and virtual absence of freeglutarate in the product mixture.

The second critical observation was the completeinability of NH3

+–PEG–COO− to bind to QAESephadex A-25 around neutral pH, i.e. only PEGmolecules with a net negative charge were retainedon the gel in distilled water. For this reason, all re-maining NH2-groups in partially glutarylated PEGdiamine were blocked with Boc2O, and the excessof Boc2O was reacted with aqueous CH3–NH2,before the mixture was applied to the anion ex-change column. CH3–NH–Boc, Boc–NH–PEG–NH–Boc, X–PEG–NH–Boc, and X–PEG–X were eluted inthe flow-through, while the desired product Boc–NH–PEG–COO− and the byproduct X–PEG–COO− wereretained on the column and subsequently elutedwith 20 mM Tris–HCl. The small amount of bound−OOC–PEG–COO− and possible traces of glutaratewere eluted with concentrated NaCl and the columnwas re-equilibrated with water for the second columnstep.

Meanwhile the mixture of Boc–NH–PEG–COOHand X–PEG–COOH was quantitatively deprotected in98% formic acid and re-applied to the ion-exchangecolumn in distilled water. Now the byproductX–PEG–COO− was again retained on the column


while the desired product NH3+–PEG–COO− wasrecovered in the flow-through. This product was per-fectly pure in the sense that every PEG molecule hadone NH2-group and one COOH, as previously foundfor NH2–PEG–COOH prepared from the same start-ing material by a less convenient synthetic route[23].

3.2. Long heterobifunctional crosslinkers forcovalent tethering of probe molecules to AFM tips

Fig. 2shows the collection of long crosslinkers pre-pared from the key intermediate NH2–PEG–COOHwith n ∼ 18 ethylene glycol units (PEG 800). In a pre-vious study[23], biotin–PEG–NHS was synthesizedin this way and attached to NH2-functionalized AFMtips. In parallel, avidin was irreversibly adsorbed to amica substrate. Tip and substrate were mounted in theAFM in which single avidin–biotin unbinding eventswere observed in 20–25% of all force–distance cycles(similar to the solid trace inFig. 5A). The specificityof single avidin–biotin interactions was corrobo-rated by the characteristic unbinding force (40 pN at1.4 nN s−1 loading rate) and by the efficient blockingin the presence of free d-biotin. In conclusion, avidinon mica and biotin–PEG tentacles on AFM tips wereshown to represent a convenient start-up and test sys-tem for single molecule recognition force microscopyand spectroscopy.

The next three crosslinkers shown inFig. 2(mal–PEG–NHS, VS–PEG–NHS, and PDP–PEG–NHS)closely resemble well-knownshort heterobifunctionalcrosslinkers, such as LC-SMCC and SPDP, except forthe long PEG 800 spacer elements. In fact, the NHSester function of these short crosslinkers were reactedwith the NH2-terminus of NH2–PEG–COOH, takingcare to quantitatively derivatize all NH2-groups. Af-ter removal of all small molecules by gel filtrationin distilled water on Sephadex G-25, the COOH ter-minus of PEG was converted into the amino-reactiveNHS ester function which provides for amide bondformation with NH2-functionalized AFM tips (seeFig. 1). The other end groups of mal–PEG–NHS,VS–PEG–NHS, and PDP–PEG–NHS (seeFig. 2) arespecific thiol-reactive functions which allow to couplea probe molecule via its free thiol.

In all AFM studies published since 1998 from thisand from collaborating labs, PDP–PEG–NHS synthe-sized by the new method described above has been

used for tip-probe linking. Most frequently, antibod-ies or other proteins were linked by the procedureshown inFig. 1. This means that the antibodies had tobe modified with mercaptopropionyl residues[8,24]before the final coupling step. In one study, a half an-tibody molecule was attached via its free sulfhydryl inthe hinge region[17]. Even more straightforward wasthe coupling of a peptide with a single cysteine residue(His6-Gly-Cys) via its free sulfhydryl[15,16]. In oneinstance, however, the PEG–PDP tentacle on the tipwas reduced by DTT and the resulting PEG–SH wassubsequently alkylated with iodoacetamidophlorizinto yield an AFM probe with high affinity for theNa/glucose cotransporter in brush border membranes[18].

In the present study, the additional new crosslinkersmal–PEG–NHS and VS–PEG–NHS were synthesizedfor the irreversible linking of SH-containing probemolecules to AFM tips. The vinylsulfone and themaleimide group are known for selective couplingto free thiols [27,40,41]. It therefore appears thatmal–PEG–NHS and VS–PEG–NHS are valuable al-ternatives to PDP–PEG–NHS when irreversible thiolcoupling is needed instead of disulfide bond forma-tion. What remains to be tested, is the preservationof the maleimide or the vinylsulfone group when theNHS ester terminus is coupled to NH2-functionalizedtips in chloroform/TEA solution. In a previous studywe found no indication that the maleimide group ofLC-SMCC reacts with a soluble primary amine inchloroform/TEA[27] but this cannot be extrapolatedto AFM tips where other nucleophilic groups may bepresent, as well. A model study on this topic is underway in order to provide reliable information to recog-nition force microscopists who, for obvious reasons,stick to well characterized AFM crosslinkers only.

3.3. Reversible tethering of His6-tagged probemolecules to AFM tips

Whenever PEG 800 spacers are used for the linkingof probe molecules to measuring tips, the PEG acts as asoft elastic band, thus the tip can be lifted≤8 nm fromthe substrate[23] before the specific recognition bondbetween probe and target molecule is ruptured. A spe-cial study has been devoted to measurement and anal-ysis of the nonlinear (non-Hook) elasticity of the PEG800 chain on the AFM tip[16]. These measurements


were based on a model system in which the noncova-lent bond between NTA·Ni2+ groups on the substrateand His6 groups on the tip could be ruptured andreformed for an indefinite number of cycles[15].

The high rupture force of the NTA·Ni2+–His6bond (160 pN at a loading rate of 10 nN s−1 [15])came as a pleasant surprise since many biologicalprobe-target interactions rupture at much lower forces[14]. In the same year, two more AFM studies onbond rupture between single tip-bound His6 peptidesand substrate-bound NTA·Ni2+ residues came outwhich also indicated high mechanical strength of theNTA·Ni2+–His6 bridge[42,43]. These findings pavedthe way for reversible PEG-probe molecule linkingon AFM tips via NTA·Ni2+–His6 bonds (seeFig. 4),in other words to use the “metal chelating microscopytip as a new toolbox for single molecule experimentsby AFM” [43].

The first successful example of tip-PEG-probe link-ing via the NTA·Ni2+–His6 bond is shown inFig. 4.The NH2-functionalized tip was derivatized withmercaptopropionyl groups to which PDP–PEG–NTAwas coupled via disulfide linkage. In the presenceof Ni2+, His6-tagged MBP-VLDLR1-3 was tightlybound to the NTA·Ni2+ function. Human VLDLRserves as a receptor for human rhinovirus serotype2 (HRV2) and from binding studies using differentrecombinant soluble fragments of VLDLR the bind-ing site has been localized to the ligand bindingrepeats 2 and 3[33,35]. Fortunately, Ni2+ was alsohelpful to immobilize the virus particles on ultraflatmica by simple electrostatic adsorption. The viruses

�

Fig. 4. Flexible tethering of a His6-tagged probe molecule to anAFM tip. (A) The amino groups on the tip (seeFig. 1) werederivatized with mercaptopropionyl residues by reaction with SATPin chloroform/TEA, followed by cleavage of theS-acetyl groupswith hydroxylamine. The resulting free thiol on the AFM tipwas then reacted with the pyridyldithio group of PDP–PEG–NTA,yielding a stable disulfide bond. The NTA function was chargedwith Ni2+ and an ectodomain fragment of the human VLDLR wasspecifically bound via its His6-tag. MBP-VLDLR1-3 contained thethree N-terminal ligand binding repeats of human VLDLR fusedat the N-terminus to maltose-binding protein and at the C-terminusto a hexa-His-tag. (B) Human rhinovirus particles (HRV2) wereelectrostatically bound to freshly cleaved muscovite mica in 5 mMNi2+, yielding regular arrays of viruses. The AFM image showsa surface of 130�m × 130�m.

were deposited in regular arrays, as can be seen inFig. 4.

The specific recognition of tip-linked MBP-VLDLR1-3 by the virus protein was measured inforce–distance cycles as shown inFig. 5A. In the ap-proach phase (dotted trace) the cantilever with the tipwas lowered towards the substrate at a linear rate of


Fig. 5. The unbinding force of the specific interaction betweenmica-bound human rhinovirus 2 and a single tip-linked VLDLR1-3molecule (compareFig. 4). The distance between the AFM tipand the mica substrate (abscissa in panels A and B) was scannedat a linear rate of 60 nm s−1 and the force of cantilever bending(ordinate in panels A and B) was recorded during the approach(dotted lines) and retraction (solid lines). At the spring constantof 35 pN nm−1 the linear scan rate of 60 nm s−1 corresponded to alinear force velocity of 2.1 nN s−1. Panel A was recorded in bufferB which contained 0.1 mM Ni2+ and 1 mM Ca2+ in addition toNaCl and Tris–HCl. The negative deflection in the retrace (solidtrace in panel A) corresponded to a specific unbinding event with38 pN rupture force. Panel B was recorded after 30 min incubationin buffer C which contained 100 mM EGTA besides NaCl andTris–Cl. Panel C shows the probability density function (pdf)of the unbinding forces measured in 40 retraces with a specificunbinding event as exemplified in panel A. The most probableunbinding force (peak maximum) was 42± 4 pN.

60 nm s−1. At the distanced = 0 nm, the apex of thetip made contact with the hard mica substrate. Furtherlowering of the tip led to an upward deflection of thecantilever, expressed in terms of bending force (y-axisin Fig. 5A) on the basis of the known spring constantof the cantilever (35 pN nm−1). Upon retraction of thecantilever holder (solid trace inFig. 5A), the bendingof the cantilever is relaxed untild = 0 nm is reachedagain. No force is observed during the next∼10 nmof retraction but then a progressive downward deflec-tion is seen which reflects the specific adhesion of asingle MBP-VLDLR1-3 molecule to a virus particle.The slope of the downward deflection is flatter thanthe slope atd < 0 nm, due to the higher elasticity ofthe PEG chain as compared to the cantilever itself. Atd ∼ 16 nm, the specific adhesion is ruptured and thecantilever jumps into the resting position. The magni-tude of the jump corresponds to the unbinding force(38 pN). A representative force distribution of 40 dif-ferent force–distance cycles with specific unbindingevents is shown inFig. 5C. The peak maximum wasfound at an unbinding force of 42± 4 pN.

The specificity of the interaction was demonstratedby its sensitivity to EGTA. After addition of EGTA,no specific adhesion events were seen in subsequentforce–distance cycles, as exemplified byFig. 5B.EGTA was expected to abolish specific binding intwo ways. On the one hand, Ca2+ ions are essentialfor the structural integrity of MBP-VLDLR1-3[35].On the other hand, Ni2+ ions are required for main-taining the NTA·Ni2+–His6 bridge between tip andMBP-VLDLR1-3. While it could not be discrimi-nated which divalent cation was first removed on thetime scale of the AFM experiment, the EGTA block(as in Fig. 5B) indicated that the specific unbindingevents (as inFig. 5A) were caused by interaction ofMBP-VLDLR1-3 with the virus particles.

A unique feature of virus recognition by tip-tetheredMBP-VLDLR1-3 is the relaxed cantilever position(zero force) at 0≤ d ≤ 10 nm in the solid traceof Fig. 5A. This must be attributed to the interplaybetween the height of the virus itself, the lengthof the PEG 800 crosslinker, and the position ofPEG 800 attachment on the AFM tip. There are 60symmetry-related receptor attachment sites distributedover the icosahedral virus surface, thus, binding siteson the side and on the top of the particle can be reachedby those PEG–VLDLR tentacles also which are


anchored higher up on the side of the AFM tip. Thisexplains the nearly 10 nm tip–substrate distance atthe begin of the downward deflection (see solid tracein Fig. 5A). Shorter and longer distances were alsoobserved in other force–distance cycles but the 10 nmdistance shown inFig. 5A was most frequent.

Fortunately, the NTA·Ni2+–His6 linkage betweenMBP-VLDLR1-3 and the PEG tentacles on the tipwas quite stable; only after 1–2 h, the chance to see aspecific recognition event in one force–distance cyclebecame noticeably lower, indicating a surprisinglyslow dissociation of His6-tagged probe moleculesfrom the tip. From this follows that His6-taggedprobe molecules to be used in recognition force mi-croscopy/spectroscopy can reliably be tethered toAFM tips with PEG–NTA tentacles by the procedureshown inFig. 4.

It should be noted that the unbinding experimentsshown inFig. 5 were done at various loading rates(force increase per second), yielding a linear de-pendence of unbinding force upon the logarithm ofloading rate (data not shown) which is typical fora single energy barrier[44,45]. Moreover, the samemethod was successfully used in our lab to recon-stitute the biospecific interaction between the smallGTPase Ran and the nuclear import receptor importin�1 in the AFM, using PDP–PEG–NTA to tetherHis6-tagged Ran to the AFM tip (compareFig. 4),while importin �1 was linked to functionalized micavia short (∼2 nm) spacers[46]. Here, again, the lineardependence of unbinding force upon loading rate wasobserved, and the unbinding force at a given loadingrate was switched from high to low when bound GTPwas hydrolyzed to GDP by the small GTPase Ran.Such large, consistent data sets can justly be taken asverification of the coupling scheme shown inFig. 4even though our usual macroscopic marker enzymeassay of coupling site density[13,23] could not bedone, due to lack of a His6-tagged marker enzyme.

3.4. Comparison of covalent versus noncovalenttethering of probe molecules to AFM tips

Covalent linking of probe molecules to AFM tipsis very straightforward when the probe molecule al-ready contains a free sulfhydryl (compareFig. 1),as is the case with a number of native proteins, withgenetically engineered cysteine mutants, or with prop-

erly synthesized peptides, oligonucleotides or othersmall molecules. In any case, special care must betaken to prevent sulfhydryl oxidation since protectiveagents like DTT or 2-mercaptoethanol cannot be usedfor obvious reasons. Proteins without free sulfhydrylgroups, however, need to be pre-derivatized withSATP to introduceS-acetyl-protected mercaptopropi-onyl groups which can be coupled to PDP groups afterde-acetylation with hydroxylamine (seeFig. 1) [8,24].The pre-derivatization requires at least 0.1–0.2 mgof protein, preferably at≥1 mg ml−1 protein concen-tration. Free SATP must rigorously be removed bygel filtration which causes sample dilution, and thealiquots of the diluted protein may denature uponrefreezing.

No such limitations are experienced when His6-tagged proteins are tethered to AFM tips. His6 is amost popular tag allowing for purification of an over-expressed protein in a single step on an NTA–Ni2+column. The same tag now provides for a long-lastinglink to PEG–NTA–Ni2+ tentacles on AFM tips, asdepicted inFig. 4, thereby eliminating the need forchemical pre-derivatization of the probe molecule. Af-ter purification, the His6-protein can immediately befrozen in small aliquots and only one aliquot at a timeneeds to be thawed and incubated with the function-alized AFM tip. Due to the stability of the His6-tag,even reuse of the same protein aliquot should be pos-sible in case of proper storage conditions, whereasthe free thiol needed for covalent coupling (Fig. 1) issensitive to oxidation.

As a consequence, noncovalent coupling (Fig. 4) isthe method of choice if the probe molecule is alreadyavailable as a His6-tagged protein. The same applieswhen the protein has been cloned and extensive recog-nition force studies are intended. In contrast, covalentcoupling (Fig. 1) is much more appropriate for shortterm studies with untagged proteins or other probemolecules. Thus, the two methods nicely complementeach other, and the tools for both methods are nowavailable, as introduced in the present study.

Acknowledgements

We are indebted Prof. Wolfgang Buchberger, Prof.Heinz Falk, and Prof. Norbert Müller for use of spec-trometer facilities. This work was supported by the


Austrian Research Funds (projects P-12801, P12802,P-14549, and P-15295).

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