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Analytica Chimica Acta 497 (2003) 101–114 Heterobifunctional crosslinkers for tethering single ligand molecules to
Analytica Chimica Acta 497 (2003) 101–114 Heterobifunctional crosslinkers for tethering single ligand molecules to

Analytica Chimica Acta 497 (2003) 101–114

Analytica Chimica Acta 497 (2003) 101–114 Heterobifunctional crosslinkers for tethering single ligand molecules to

Heterobifunctional crosslinkers for tethering single ligand molecules to scanning probes

Christian K. Riener a , Ferry Kienberger a , Christoph D. Hahn a , Gerhard M. Buchinger a , Innocent O.C. Egwim a , Thomas Haselgrübler a , Andreas Ebner a , Christoph Romanin a , Christian Klampfl b , Bernd Lackner b , Heino Prinz c , Dieter Blaas d , Peter Hinterdorfer a , Hermann J. Gruber a,

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

b 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, Germany d 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 probe specific interactions of cognitive molecules on the single molecule level. It allows insights to be gained into interaction potentials and kinetic barriers and is capable of mapping interaction sites with nm positional accuracy. These applications require a ligand to be attached to the AFM tip, preferably by a distensible poly(ethylene glycol) (PEG) chain between the measuring tip and the ligand molecule. The PEG chain greatly facilitates specific binding of the ligand to immobile receptor sites on the sample surface. The present study contributes to tip-PEG-ligand tethering in three ways: (i) a convenient synthetic route was found to prepare NH 2 –PEG–COOH which is the key intermediate for long heterobifunctional crosslinkers; (ii) a variety of heterobifunctional PEG derivatives for tip-PEG-ligand linking were prepared from NH 2 –PEG–COOH; (iii) in particular, a new PEG crosslinker with 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; Boc 2 O, O,O -di-tert-butyldicarbonate; biotin–PEG–NHS, see Fig. 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, see Fig. 2; MBP-VLDLR1-3, fusion product of maltose-binding protein with the three N-terminal repeats of the very-low density lipoprotein receptor; NH 2 –PEG–NH 2 , O,O -bis(2-aminopropyl)poly(ethylene glycol) 800; NH 2 –PEG–COOH, N-glutaryl derivative of NH 2 –PEG–NH 2 ; 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, see Fig. 2; PDP–PEG–NTA, see Fig. 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, see Fig. 2; X–PEG–NH 2 , impurity of NH 2 –PEG–NH 2 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: hermann.gruber@jku.at (H.J. Gruber).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.aca.2003.08.041

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His 6 -tagged protein molecules to AFM tips via noncovalent NTA–Ni 2+ –His 6 bridges. The new crosslinker was applied to link a recombinant His 6 -tagged fragment of the very-low density lipoprotein receptor to the AFM tip whereupon specific docking to the capsid of human rhinovirus particles was observed by force microscopy. In a parallel study, the specific interaction of the small GTPase Ran with the nuclear import receptor importin 1 was studied in detail by SMRFM, using the new crosslinker to link His 6 -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); Heterobifunctional crosslinker

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 study biospecific interactions on various scales of size and

time (see [1] for a review). Very detailed results are obtained by single molecule recognition force mi- croscopy (SMRFM) which can measure interaction forces on the single molecule level. The basic tool for SMRFM is a measuring tip whose apex has been functionalized with one or few probe molecules that can recognize a specific type of target molecules. In

a number of laboratories, AFM tips have been coated

with probe molecules which were either strongly adsorbed [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 between the probe molecule and the apex of the measuring tip affords significant technical improvements: (i) the PEG 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) the probe molecule escapes the danger of being crushed between tip and substrate during hard contact; (iv) most importantly, the soft and nonlinear elasticity of the PEG tether allows to clearly distinguish between unspecific and receptor-specific binding of the tip. The nonlinear elasticity of the 6–8 nm long PEG spacer upon stretching up to the moment of receptor–ligand bond rupture has thoroughly been characterized in

a preceding AFM study [16] and quantitatively ana-

lyzed according to theoretical models [20–22]. The standard procedure for tip-PEG-probe linking is depicted in Fig. 1. In the first step, the amino- functionalized AFM tip [8,23] is reacted with the NHS-ester terminus of the PEG crosslinker. Usually

about 1–3 PEG tentacles are attached which can reach below the apex of the AFM tip [13,23]. In the second step, the pyridyldithio group at the outer end of PEG reacts with a free thiol group on the probe molecule, yielding a stable disulfide, while 2-mercaptopyridine is released which immediately tautomerizes into 2-thiopyridone [24,25]. The original synthesis [24] of the long crosslinker in Fig. 1 had several drawbacks. It required large amounts of the expensive reagent SPDP in the first

large amounts of the expensive reagent SPDP in the first Fig. 1. Standard procedure for the

Fig. 1. Standard procedure for the tethering of single antibody molecules to AFM tips. Amino groups are generated on the tip by one of three characterized procedures [23]. In step 1, these amino groups react with the NHS ester functions of the PEG crosslinker molecules, yielding stable amide bonds. In step 2, the pyridyldithio group on the outer end of PEG reacts with a free thiol group on the probe molecule, resulting in a stable disulfide bond while 2-mercaptopyridine is released.

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step, yields were moderate, and the synthetic route was not adaptable to the synthesis of similar AFM crosslinkers with other coupling functions. These problems were overcome by a new synthetic route in which the central element (NH 2 –PEG–COOH, see Fig. 2) was synthesized first [23]. As can be seen from Fig. 2, NH 2 –PEG–COOH is the key intermediate for

Fig. 2 , NH 2 –PEG–COOH is the key intermediate for Fig. 2. NH 2 –PEG–COOH

Fig. 2. NH 2 –PEG–COOH as a precursor to various long heter- obifunctional crosslinkers. Biotinylation of NH 2 –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-reactive functions were attached to the NH 2 -group of NH 2 –PEG–COOH, followed by activation of the terminal carboxyl groups as NHS ester (b). In case of PDP–PEG–NHS, the crosslinker was further reacted with lys-DA to introduce the NTA function (f), useful for the linking of His 6 -tagged probe molecules (see Fig. 4).

linking of His 6 -tagged probe molecules (see Fig. 4 ). Fig. 3. Synthesis of the

Fig. 3. Synthesis of the key intermediate (NH 2 –PEG–COOH) for heterobifunctional PEG derivatives. (a) 0.2 equivalents of glutaric anhydride; (b) excess of Boc 2 O; (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 NH 2 –PEG–COOH because in the first and third chromatographic step it was tricky to achieve high purity without sacrificing too much product. In the present study, NH 2 –PEG–COOH has been synthesized by a new method (see Fig. 3) which re- quires only two chromatographic steps in water and gives pure product in high yield. Furthermore, a vari- ety of crosslinkers for tip-PEG-probe conjugation have been synthesized, including a new PEG derivative with an NTA group for noncovalent linking of His 6 -tagged proteins (see Fig. 2). The use of the latter is exempli- fied by tethering of a His 6 -tagged ectodomain frag- ment of the VLDLR to an AFM tip and measurement of specific adhesion events with mica-bound human rhinovirus particles see Figs. 4 and 5.

2. Materials and methods

2.1. Materials

Analytical

grade

materials

were

used

if

they

were

commercially

available.

The

starting

mate-

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rials were of the highest available purity. Unless stated otherwise, buffer components and organic solvents, as well as acetic acid, ammonia solution (32% in water), DCC, D 2 O, formic acid, hydroxy- lamine hydrochloride, LiChroprep RP-18 gel, methy- lamine (40% in water), trifluoroacetic acid, were obtained from Merck (Germany). DIEA was pur- chased from Aldrich (Austria). Sephadex G-25M and QAE-Sephadex A-25 were obtained from Amersham Biosciences (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 supplied by Christiane Gröpl (Austria). LC-SMCC was ob- tained from Molecular Biosciences (Colorado). Si 3 N 4 measuring tips were bought from Park Scientific (Sun- nyvale, CA). Boc 2 O, CDCl 3 , 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

deprotection), all other components were visualized in iodine vapor.

2.4. Synthesis of NH 2 –PEG–COOH

In a 250-ml round bottomed flask, O,O -bis(2- aminopropyl)poly(ethylene glycol) 800 (9.1 g, 9.6 mmol) was dissolved in chloroform (125 ml) and stirred under an argon atmosphere. The solution was cooled in liquid nitrogen until the solution began to freeze (35 C solution temperature). The liquid nitrogen 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 argon atmosphere. The temperature rose to +10 C within

the first hour and to +20 C within the second hour. After 3 h, the solvent was evaporated on the rotavap at 30 C bath temperature. The honey-like product was dissolved in methanol (75 ml) and stirred in

a 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 added

[26].

with stirring. Subsequently, an argon-bubbled solution

 

of

Boc 2 O (4.36 g, 20 mmol) in methanol (7.5 ml) was

2.2.

Buffers

added dropwise. The completion of the reaction was

Buffer A contained 100 mM NaCl, 50 mM NaH 2 PO 4 , and 1 mM EDTA (pH 7.5 adjusted with NaOH). Buffer B contained 150 mM NaCl, 0.1 mM NiCl 2 , 1 mM CaCl 2 , and 50 mM Tris base (pH 7.6 adjusted with HCl). Buffer C contained 150 mM NaCl and 100 mM EGTA (pH 7.6 adjusted with Tris base). Buffer D contained 50 mM Tris base and 5 mM NiCl 2 (pH 7.6 adjusted with HCl). PBS contained 150 mM NaCl and 5 mM NaH 2 PO 4 (pH 7.4 ad- justed with NaOH). Hydroxylamine reagent (500 mM hydroxylamine·HCl, 25 mM EDTA, pH 7.5) was prepared as described [24].

checked by TLC in chloroform/methanol/acetic acid (80/20/2, v/v/v). The excess of Boc 2 O was quenched by 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 was cooled to 0 C and the solvent was removed by ro- tary evaporation. The residue was dried at 1–10 Pa for 3 h, dissolved in water (110 ml) and the pH was adjusted to 7.5 with dilute NaOH. The solution was frozen to the walls of a 2-l flask and lyophilized to remove excess of methylamine. The dry residue was dissolved in water (200 ml final volume), the pH was readjusted to 7.5, and the solution was loaded at a flow of 4 ml min 1 on an anion exchange column

2.3.

Thin layer chromatography

(QAE Sephadex A-25, 5 cm diameter, 17.5 cm bed

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, or acetic acid, respectively. Amino groups were specif- ically stained with ninhydrin (Boc–NH-groups were also stained after 1 min at 100 C, due to thermal

height, chloride form, stored in 20% ethanol, precon- ditioned with >2 bed volumes of distilled water at a flow 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 derivatives were eluted, as detected by spotting of eluate on TLC plates and developing in eluent I. Elution was contin- ued with 20 mM Tris (adjusted to pH 7.5 with HCl,

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

105

1460 ml, fractions # 8–22). The column was regen- erated from dicarboxylic acids by washing with 5 M NaCl, 50 mM Tris (pH 7.5 adjusted with HCl, 700 ml) and re-equilibrated in distilled water (1000 ml) for the second column step. Fractions # 11–18 were pooled and taken to dryness by rotary evaporation without heating, using a strong vacuum source. The dry residue was dissolved in 50 ml of 6 M NaCl and extracted with chloroform (1 × 50 ml, 2 × 25 ml). The combined organic phases were dried with Na 2 SO 4 , filtered, and taken to dryness. The residue was dried at 1–10 Pa for 3 h, yielding 1.38 g of a honey-like product which was split into two portions. The 1 H NMR spectrum showed the expected singlet for the Boc group at 1.43 ppm in addition to the other signals of NH 2 –PEG–COOH. For cleavage of the Boc-group, one portion (741 mg) of the above product was dissolved in 2.6 ml of water and 17.3 ml of formic acid was added. After 3 h of stirring at RT, 17.3 ml of toluene was added and 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. The honey-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 and re-equilibrated anion exchange column (see above). The column was eluted with water (600 ml) while collecting 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 with water (1000 ml) and either reused or stored in 20% ethanol. Fractions # 8–15 were pooled and taken to dryness by rotary evaporation without heating, us- ing a strong vacuum source. The dry residue was dissolved in 20 ml 6 M NaCl and extracted with chlo- roform (4 × 15 ml). The combined organic phases were dried with Na 2 SO 4 , filtered, taken to dryness, and the residue was dried at 1–10 Pa for 3 h, yielding 0.534 g (0.50 mmol) of a honey-like product which was pure by TLC (R I f = 0.27–0.53, R II = 0.23–0.48,

R

1 H NMR (200 MHz, CDCl 3 ) δ: 1.06–1.2 (10H, m, NH 2 –CH<(CH 3 )–CH 2 –O and NH 2 –CH<(CH 2 – CH 3 )–CH 2 –O of PEG), 1.94 (2H, quin, J = 7 Hz,

CO–CH 2 –CH 2 –CH 2 –CO, position in glu), 2.26 (2H, t, CH 2 –CH 2 –COOH), 2.36 (2H, t, J = 7 Hz, CH 2 –CH 2 –CO–NH), 3.35–3.54 (6H, m,

f

III

f

= 0.26–0.51).

N–CH <(CH 3 )–CH 2 –O and N–CH <(CH 2 –CH 3 )– 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, CH 2 –CO–NH –CH<(C)–CH 2 –O), 7.26 (s, CDCl 3 ), 7.88 (3H, broad s, NH 3 + ). In the preceding study it has been shown that 50% of the termini in com- mercial NH 2 –PEG–NH 2 were 2-aminobutyl and not 2-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 NH 2 –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 under argon was continued. For monitoring the reaction by TLC, a small aliquot was evaporated at 1–10 Pa and redissolved in a drop of chloroform before spotting. After 30 min, traces of ninhydrin-reactive

NH 2 –PEG–COOH were still visible while after

60 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 LiChroprep RP-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 product was 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 containing 0.1% 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 and dried at 1–10 Pa overnight, yielding 21 mg (15 mol

mal–PEG–COOH) of viscous product which was pure

by TLC (R f I 0.36–0.57, R II 0.56–0.70, R III 0.56–0.67).

f

f

1 H NMR: (500 MHz, in CDCl 3 , 1-dim 1 H and 2-dim 1 H– 1 H– and 13 C– 1 H–COSY; [29]) δ

(ppm): 0.99 (2H, dq, 13/3 Hz, axial ring positions

3 and 5 [27]), 1.08–1.18 (10 H, m, NH 2 –CH<

(CH 3 )–CH 2 –O and NH 2 –CH<(CH 2 –CH 3 )–CH 2 –O of PEG), 1.33 (2H, quin, 7.5 Hz, C–CH 2 –C position

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

in aminohexanoyl), 1.42 (2H, dq, 13/3 Hz, axial ring positions 2 and 6), 1.50 (2H, quin, 7.5 Hz, C–CH 2 –C position 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, CH 2 –CH 2 –CH 2 , 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 –CH 2 –CH 2 position in glu), 2.36–2.41 (2H, m, CH 2 –CH 2 –CH 2 –COOH position in glu), 3.19–3.26 (2H, m, 7.5 Hz, O–CO–CH 2 –CH 2 position ε in aminohexanoyl),

mixture. After stirring at RT overnight, the solution was filtered from dicyclohexylurea and the filtrate was evaporated, yielding 7.9 mg of viscous product which

was pure by TLC (R f I 0.69–0.85, R II

0.65–0.71, R III

f

f

0.71–0.81), except for contamination with free NHS (R f I 0.02–0.13). The product gave the same NMR spectrum as the free carboxylic acid form, except for the additional singlet at 2.86 ppm (NHS residue) and the typical shift of the -CH 2 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

3.36

(2H, d, 7 Hz, >N–CH 2 –C< bridge between

NH 2 –PEG–COOH (49 mg, 45 mol) was dis-

maleimide and the ring), 3.40–3.58 (6H, m, N–CH <

solved in 1.5 ml of chloroform and stirred under an

(CH 3 )–CH 2 –O and N–CH <(CH 2 –CH 3 )–CH 2 –O of PEG), 3.60–3.70 (72H, s, O–CH 2 –CH 2 –O,

argon atmosphere and TEA (21 l, 150 mol) was added. SPDP (21 mg, 65 mol) was dissolved in

18 ethylene glycol units [23]), 4.09–4.14 (2H, m, CH 2 –CO–NH –CH 2 of both PEG termini), 6.69 (2H,

chloroform (1 ml) and added to the mixture under continued stirring under argon. After 10 min, TLC

s,

N–CH =CH –N of mal), 7.26 (s, CDCl 3 ). Due to the

showed complete conversion of NH 2 –PEG–COOH

long T 1 relaxation time of the olefinic hydrogen atoms,

into PDP–PEG–COOH and the solvent was removed.

a

25 s delay time between pulses was required to

The residue was thoroughly suspended in buffer A

obtain a correct integral for the maleimide group [30].

(2 ml). Undissolved SPDP was removed by cen-

13 C NMR: (500 MHz, in CDCl 3 , 1-dim 13 C and 2-dim 13 C– 1 H–COSY; [29,31] δ (ppm): 17.6 (16.6) (N–CH<(CH 2 )–CH 3 of PEG-2(3)-amino-butylterm) 21.0 (C–CH 2 –C position in glu), 25.0 (C–CH 2 –C position in LC), 26.2 (C–CH 2 –C position in LC), 28.8 (positions 2 and 6 in cyclohexane), 29.0 (C–CH 2 –C position in LC), 29.9 (positions 3 and 5 in cyclohexane), 32.9 (C–CH 2 –CO–O position

trifugation (10,000 g, 10 min) and by filtering the supernatant (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 argon was continued for 10 min. After acidification to pH 4.5 with acetic acid, 22.5 l (225 mol) of divinylsul- fone (harmful!) was pipetted into the solution and the pH was gradually raised to 7 with 10% Na 2 CO 3 with

in

glu), 35.2 (N–CO–CH 2 –C position in glu), 36.1

continued argon bubbling. After 2 h the reaction was

(C–CH 2 –CO–N position in LC), 36.4 (position 4 in cyclohexane), 39.0 (O–CO–CH 2 –C position ε in LC),

stopped by acidification to pH 4.5 with 1 M HCl. The mixture was diluted to 4 ml with water and loaded on

43.7

(>N–CH 2 –C< bridge between maleimide and

a Sephadex G-25F column (2.5 cm × 44 cm, condi-

cyclohexane), 45.2 (position 1 in cyh), 45.3 (N–CH<

tioned in distilled water). The column was eluted with

of PEG-2(3)-aminobutylterm) 70.5 (O–CH 2 CH 2 –O 18 units of PEG), 75.0 (73.9) (N–CH–CH 2 –O of PEG-2(3)-aminobutylterm), 77.0 (CDCl 3 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-

distilled water at a flow of 2.7 ml min 1 while collect- ing 8-ml size fractions. The column was regenerated with 160 ml of 3 M NaCl and either washed with wa- ter for another use or stored in a solution containing 40 mM Tris base and 5 mM sodium azide. Fractions containing the product (# 12–19) were pooled and

etate

(160 l) and 1.4 mg of NHS (12 mol) was

saturated with NaCl. The product was extracted into

dissolved in 120 l of ethyl acetate and 90 l thereof

chloroform (4 × 30 ml), the combined organic phases

were

added to mal–PEG–COOH. Then, 2.5 mg of

were dried over Na 2 SO 4 , filtered, and the filtrate was

DCC

was also dissolved in 120 l of ethyl acetate,

evaporated. Overnight drying at 1–10 Pa gave 30 mg

and 90 l thereof were pipetted into the reactions

of viscous product (24 mol VS–PEG–COOH)

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

107

which was pure by TLC (R I f 0.24–0.45, R II 0.52–0.72,

f

0.53–0.69). It was dissolved in dry ethyl acetate

(0.4 ml), NHS (52.6 mg, 24 mol) was added and completely dissolved with stirring. DCC (29.3 mg, 24 mol) was added and the solution was stirred at RT overnight. Dicyclohexylurea was filtered off and the filtrate was evaporated, yielding 25 mg of viscous product (18 mol VS–PEG–NHS) which was pure by TLC (R f I 0.55–0.68, R II 0.56–0.65, R III 0.56–0.77).

R III

f

f

f

1 H NMR: (500 MHz, 1 H-spectrum, 2-dim 1 H– 1 H– COSY in CDCl 3 ) (ppm): 1.03–1.24 (10 H, m, NH 2 –CH<(CH 3 )–CH 2 –O and NH 2 –CH<(CH 2 – CH 3 )–CH 2 –O of PEG), 2.11 (2H, quin, J = 7 Hz,

CH 2 –CH 2 –CH 2 , position in glu), 2.28 (2H, t, J = 7 Hz, NH–CO–CH 2 –CH 2 , position in glu),

2.45 (2H, t, J = 7 Hz, NH–CO–CH 2 –CH 2 –S),

2.69 (2H, t, J = 7 Hz, CH 2 –CH 2 –CO–NHS), 2.78–

2.97 (4H, m, NH–CO–CH 2 –CH 2 –S and S–CH 2

CH 2 –SO 2 ), 2.85 (4H, s, NHS residue), 3.21–3.33 (2H, m, S–CH 2 –CH 2 –SO 2 ) 3.34–3.56 (6H, m, N–CH <(CH 3 )–CH 2 –O and N–CH <(CH 2 –CH 3 )– CH 2 –O of PEG), 3.64 (72H, s, O–CH 2 –CH 2 –O, 18 ethylene glycol units [23]), 4.03–4.19 (2H, m, CH 2 –CO–NH –CH 2 of both PEG termini), 6.21 (1H, d 1 , 3 J cis = 9.8 Hz, SO 2 –CH=CH cis H), 6.45 (1H, d 1 , 3 J trans = 16.6 Hz, SO 2 –CH=CHH trans ), 6.70 (1H, dd 1 , 3 J trans = 16.6 Hz and 3 J cis = 9.8 Hz, SO 2 –CH =CH 2 ), 7.26 (s, CDCl 3 ).

2.7. Synthesis of PDP–PEG–NHS

NH 2 –PEG–COOH (474 mg, 0.45 mmol) was dissolved in chloroform (7 ml) and stirred under argon atmosphere. TEA (0.21 ml, 1.5 mmol) and SPDP (205 mg, 0.66 mmol) were added. The reac- tion was monitored by TLC to make sure that all ninhydrin-reactive educt was derivatized with SPDP. Then, the solvent was evaporated, the residue was dried at 1–10 Pa for 3 h and thoroughly suspended in buffer A (12.5 ml). After centrifugation (20 min at 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 distilled water at a flow of 2.7 ml min 1 while collecting 8 ml fractions. The column was regenerated with 160 ml of 3 M NaCl and either re-equilibrated in water for an- other use or stored in a solution containing 40 mM Tris base and 5 mM sodium azide. Elution of the product

was detected by reductive cleavage of the PDP group with 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 l buffer A, 25 l of 0.5 M 2-mercaptoethanol was added, and A 343 was measured after 5 min. Fractions con- taining the product (# 12–17) were pooled and taken to dryness by rotary evaporation without heating, us-

ing a strong vacuum source. The residue was dried at 1–10 Pa overnight, yielding 412 mg of viscous product which was pure by TLC (R I f 0.33–0.53, R II 0.65–0.81,

f

R

III

f

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

solved in ethyl acetate (3 ml), 52.6 ml of NHS was added and completely dissolved with stirring. Then, 29.3 mg of DCC was added and the solution was stirred at RT overnight. Dicyclohexylurea was filtered off, 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 (R f I 0.40–0.55,

R

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

1 H NMR: (200 MHz, in CDCl 3 ) δ (ppm): 1.04–1.26 (10 H, m, NH 2 –CH<(CH 3 )–CH 2 –O and NH 2 –CH< (CH 2 –CH 3 )–CH 2 –O of PEG), 1.96 (2H, quin,

7.2 Hz, CH 2 –CH 2 –CH 2 , position in glu), 2.23–2.48

(4H, m(2t), NH–CO–CH 2 –CH 2 –CH

sition and in glu), 2.56 (2H, t, 6.4 Hz, NH–CO– CH 2 –CH 2 –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–CH 2 –CH 2 –S–S, position 3 in prop), 3.34–3.56 (6H, m, N–CH <(CH 3 )–CH 2 –O and N–CH <(CH 2 –CH 3 )–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, CH 2 –CO–NH –CH 2 of both PEG termini), 7.12 (1H, m(dd), position H5 of pyridyl residue), 7.26 (s, CDCl 3 ), 7.65 (2H, m(dt), position H-3,4 of pyridyl residue), 8.45 (1H, m(dd), position H6 of pyridyl residue).

2 –CO–NH po-

II

f

0.69–0.88, R III

f

0.64–0.80). The molecular struc-

2.8. Synthesis of PDP–PEG–NTA

A suspension of lys-DA (15 mg, 50 mol) in methanol (0.2 ml) was stirred under argon. Upon addition of TEA (42 l, 300 mol) the solid was dissolved. This solution was slowly dropped into a

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solution of PDP–PEG–NHS (109 mg, 80 mol) in methanol (0.75 ml) while stirring under argon atmo- sphere (1 drop min 1 ). The dropping funnel was rinsed with 0.5 ml of methanol which were added to the 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, redissolved in 10 ml of 1 mM NaH 2 PO 4 (pre-adjusted to pH 7 with NaOH) and centrifuged for 20 min at 2500 × g). The supernatant was filtered (Millex GS, 0.2 m) and loaded on an anion exchange column (QAE Sephadex A-25, 1.5 cm × 12.5 cm, chloride form, conditioned in distilled water) at a flow of 1 ml min 1 . After washing with 2 bed volumes of distilled water, the column was eluted 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 HCl while collecting 4-ml size fractions. The column was regenerated with 160 ml of 3 M NaCl, reconditioned with 3 bed volumes of water and either reused or stored in 20% ethanol. Elution of the product was detected by reductive cleavage and measurement of released 2-thiopyridone by UV absorption at 343 nm, as described above (see Section 2.7). Fractions # 18–20 were pooled, and purified on a reversed phase column (Merck LiChroprep RP-18, 1.5 cm × 5 cm), as described above for mal–PEG–COOH. Fractions con- taining the product were detected by UV absorption measurement (see above), pooled, and taken to dry- ness by rotary evaporation. The residue was dried at 1–10 Pa overnight, yielding viscous product (53.5 mg, 36 mol) which was pure by TLC (R f I 0.00–0.12,

R

1 H NMR: (500 MHz, 1 H-spectrum, 2-dim 1 H– 1 H– COSY in D 2 O) δ (ppm): 1.08–1.19 (10H, m, NH 2 – CH<(CH 3 )–CH 2 –O and NH 2 –CH<(CH 2 –CH 3 )– CH 2 –O of PEG), 1.46–1.63 (2H, m, CH 2 –CH 2 –CH 2

CH(CO)–N< position I , II in lys-DA residue) 1.60 (2H, CO–NH–CH 2 –CH 2 –CH 2 , position in lys-DA residue), 1.89 (2H, quin, 7.2 Hz, CH 2 –CH 2 –CH 2 , position in glu), 1.85–2.03 (2H, m(qd), CH 2 – CH 2 –CH(CO)–N< position I , II in lys-DA residue), 2.23–2.31 (4H, m(2t), NH–CO–CH 2 –CH 2 – CH 2 –CO–NH position and in glu), 2.71 (2H, t, 6.4 Hz, NH–CO–CH 2 –CH 2 –S–S), 3.16 (2H, t, 6.4Hz, NH–CO–CH 2 –CH 2 –S–S), 3.22 (2H, m(t), CO–NH–CH 2 –CH 2 , position ε in lys-DA), 3.38–3.59 (6H, m, N–CH <(CH 3 )–CH 2 –O and

II

f

0.00–0.20, R III 0.00–0.12).

f

N–CH <(CH 2 –CH 3 )–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×) and

CH 2 –CH (COOH)–N< of lys-DA residue), 3.95–4.15

(2H, m, CH 2 –CO–NH –CH 2 of both PEG termini), 4.80 (bs, H OD), 7.63 (1H, t(dt) 1 , position H5 of pyridyl residue), 8.11 (1H, d(dd) 1 , position H3 of pyridyl residue), 8.21 (1H, t(dt) 1 , position H4 of pyridyl residue), 8.59 (1H, d(dd) 1 , position H6 of pyridyl residue).

2.9. Imaging of HRV2 particles on mica

HRV2 viral particles were prepared and purified as described [33]. Freshly cleaved mica was incubated with 100 l of 0.1 mg ml 1 HRV2 in buffer D for 15 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 spring constant of 0.1 N m 1 . The peak-to-peak oscillation amplitude was 5 nm at 7 kHz driving frequency. The feedback was adjusted to 30% amplitude reduction.

2.10. Measurement of single unbinding events between VLDLR1-3 and HRV2

His 6 -tagged VLDLR1-3 was coupled to AFM tips (Park Scientific, Sunnyvale, CA), using a multi-step protocol. First, the tips were functionalized with ethanolamine·HCl at RT [23]. Second, these amino groups were derivatized with SATP (0.5%, w/v) and TEA (0.5%, v/v) in chloroform solution (1.5 h at RT). After washing with chloroform (3×) and dry- ing with nitrogen gas, the tip was immersed in an aqueous solution prepared from 9 volumes of buffer

A containing 3 mg ml 1 of PDP–PEG–NTA and 1

volume of hydroxylamine reagent which had been deoxygenated with argon gas. After 1 h of incuba- tion at room temperature, the NTA functions on the tips were loaded with Ni 2+ by 20 min incubation

in buffer D. The cantilevers were washed in PBS

(3×) and incubated in PBS solution containing 1 M

His 6 -tagged MBP-VLDLR1-3 (a fusion product be-

tween maltose-binding protein and the first three ligand binding repeats of the human very-low density

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109

lipoprotein receptor, prepared as described in [34]) for 40 min, followed by three washing cycles in PBS. Force-displacement measurements were performed with a Macmode PicoSPM Microscope (Molecu- lar Imaging) equipped with a commercial fluid cell and with a Molecular Imaging Macmode interface driving a Nanoscope IIIa controller (Digital Instru- ments, Santa Barbara, CA). Force–distance cycles with receptor-modified tips and virus-loaded mica were carried out in buffer B. Ca 2+ ions are required to maintain the ligand binding repeats in their native conformation. Therefore, in control experiments, the interaction between virus and MBP-VLDLR1-3 [35] was specifically eliminated by complexation with EGTA (30 min incubation in buffer C). Force–distance cycles were performed with a z-range of 100 nm and a frequency 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 as described in detail [38].

3. Results and discussion

3.1. Preparation of pure NH 2 –PEG–COOH

From Fig. 2 it can be seen that NH 2 –PEG–COOH is a key intermediate for the syntheses of many long heterobifunctional crosslinkers with a PEG spacer ele- ment. In the present study, the synthetic route depicted in Fig. 3 was found to yield NH 2 –PEG–COOH with the desired purity, i.e. each PEG molecule had exactly one NH 2 -group and one COOH, as judged from TLC, ESI-MS, and 1 H NMR. The commercial educt was O,O -bis(2-aminopro- pyl)poly(ethylene glycol) 800 which is known to have an average number of 18 ethylene glycol units and only about 1.6 NH 2 -groups per PEG chain [8,23,39], in other words, 20% of the PEG diamine was present as X–PEG–NH 2 , and 4% as X–PEG–X, “X” denot- ing some unknown terminal group which was found to be uncharged between pH 2 and 12 [23]. No econom- ical way was found to eliminate the PEG molecules with one or two “missing NH 2 -groups” [23] but fortu- nately the glutarylated derivatives of these impurities were easily removed by ion exchange chromatography on QAE Sephadex A-25 in aqueous solution. This gel has an exceptionally high charge density (0.5 mol l 1

bed volume, as compared to 0.1–0.2 mol in most other gels) and tightly binds monocarboxylate deriva- tives of PEG 800 from salt-free solution. While QAE Sephadex A-50 was found to bind a higher amount of negatively charged PEG per gram of dry gel, the A-50 gel was inferior to A-25 in practice because the bed volume of A-50 expanded by an order of magnitude in distilled water (the binding condition) as compared to 10 2 to 10 1 M salt (the desorption condition). This led to poor effective capacity of Sephadex A-50 column beds and to great technical difficulties (data not shown). One critical observation for devising the procedure in Fig. 3 was the following. The binding capacity of QAE Sephadex A-25 for PEG 800 monocarboxylates was greatly diminished when a significant fraction of dicarboxylic acid salt was present. For this reason, the cheap starting material NH 2 –PEG–NH 2 was sta- tistically derivatized with as little as 0.2 equivalents of glutaric anhydride per 2 equivalents of acidimetri- cally determined NH 2 -groups (see [23] for the acidi- metric titration) which provided for a low fraction of HOOC–PEG–COOH and virtual absence of free glutarate in the product mixture. The second critical observation was the complete inability of NH 3 + –PEG–COO to bind to QAE Sephadex A-25 around neutral pH, i.e. only PEG molecules with a net negative charge were retained on the gel in distilled water. For this reason, all re- maining NH 2 -groups in partially glutarylated PEG diamine were blocked with Boc 2 O, and the excess of Boc 2 O was reacted with aqueous CH 3 –NH 2 , before the mixture was applied to the anion ex- change column. CH 3 –NH–Boc, Boc–NH–PEG–NH– Boc, X–PEG–NH–Boc, and X–PEG–X were eluted in the flow-through, while the desired product Boc–NH– PEG–COO and the byproduct X–PEG–COO were retained on the column and subsequently eluted with 20 mM Tris–HCl. The small amount of bound OOC–PEG–COO and possible traces of glutarate were eluted with concentrated NaCl and the column was re-equilibrated with water for the second column step. Meanwhile the mixture of Boc–NH–PEG–COOH and X–PEG–COOH was quantitatively deprotected in 98% formic acid and re-applied to the ion-exchange column in distilled water. Now the byproduct X–PEG–COO was again retained on the column

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while the desired product NH 3 + –PEG–COO was recovered in the flow-through. This product was per- fectly pure in the sense that every PEG molecule had one NH 2 -group and one COOH, as previously found for NH 2 –PEG–COOH prepared from the same start- ing material by a less convenient synthetic route [23].

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

Fig. 2 shows the collection of long crosslinkers pre- pared from the key intermediate NH 2 –PEG–COOH with n 18 ethylene glycol units (PEG 800). In a pre- vious study [23], biotin–PEG–NHS was synthesized in this way and attached to NH 2 -functionalized AFM tips. In parallel, avidin was irreversibly adsorbed to a mica substrate. Tip and substrate were mounted in the AFM in which single avidin–biotin unbinding events were observed in 20–25% of all force–distance cycles (similar to the solid trace in Fig. 5A). The specificity of single avidin–biotin interactions was corrobo- rated by the characteristic unbinding force (40 pN at 1.4 nN s 1 loading rate) and by the efficient blocking in the presence of free d-biotin. In conclusion, avidin on mica and biotin–PEG tentacles on AFM tips were shown to represent a convenient start-up and test sys- tem for single molecule recognition force microscopy and spectroscopy. The next three crosslinkers shown in Fig. 2 (mal–PEG–NHS, VS–PEG–NHS, and PDP–PEG–NHS) closely resemble well-known short heterobifunctional crosslinkers, such as LC-SMCC and SPDP, except for the long PEG 800 spacer elements. In fact, the NHS ester function of these short crosslinkers were reacted with the NH 2 -terminus of NH 2 –PEG–COOH, taking care to quantitatively derivatize all NH 2 -groups. Af- ter removal of all small molecules by gel filtration in distilled water on Sephadex G-25, the COOH ter- minus of PEG was converted into the amino-reactive NHS ester function which provides for amide bond formation with NH 2 -functionalized AFM tips (see Fig. 1). The other end groups of mal–PEG–NHS, VS–PEG–NHS, and PDP–PEG–NHS (see Fig. 2) are specific thiol-reactive functions which allow to couple a probe molecule via its free thiol. In all AFM studies published since 1998 from this and 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 procedure shown in Fig. 1. This means that the antibodies had to be 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 in the hinge region [17]. Even more straightforward was the coupling of a peptide with a single cysteine residue (His 6 -Gly-Cys) via its free sulfhydryl [15,16]. In one instance, however, the PEG–PDP tentacle on the tip was reduced by DTT and the resulting PEG–SH was subsequently alkylated with iodoacetamidophlorizin to yield an AFM probe with high affinity for the Na/glucose cotransporter in brush border membranes

[18].

In the present study, the additional new crosslinkers mal–PEG–NHS and VS–PEG–NHS were synthesized for the irreversible linking of SH-containing probe molecules to AFM tips. The vinylsulfone and the maleimide group are known for selective coupling to free thiols [27,40,41]. It therefore appears that mal–PEG–NHS and VS–PEG–NHS are valuable al- ternatives to PDP–PEG–NHS when irreversible thiol coupling is needed instead of disulfide bond forma- tion. What remains to be tested, is the preservation of the maleimide or the vinylsulfone group when the NHS ester terminus is coupled to NH 2 -functionalized tips in chloroform/TEA solution. In a previous study we found no indication that the maleimide group of LC-SMCC reacts with a soluble primary amine in chloroform/TEA [27] but this cannot be extrapolated to AFM tips where other nucleophilic groups may be present, as well. A model study on this topic is under way 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 His 6 -tagged probe molecules to AFM tips

Whenever PEG 800 spacers are used for the linking of probe molecules to measuring tips, the PEG acts as a soft elastic band, thus the tip can be lifted 8 nm from the substrate [23] before the specific recognition bond between 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 PEG 800 chain on the AFM tip [16]. These measurements

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were based on a model system in which the noncova- lent bond between NTA·Ni 2+ groups on the substrate and His 6 groups on the tip could be ruptured and reformed for an indefinite number of cycles [15]. The high rupture force of the NTA·Ni 2+ –His 6 bond (160 pN at a loading rate of 10 nN s 1 [15]) came as a pleasant surprise since many biological probe-target interactions rupture at much lower forces [14]. In the same year, two more AFM studies on bond rupture between single tip-bound His 6 peptides and substrate-bound NTA·Ni 2+ residues came out which also indicated high mechanical strength of the NTA·Ni 2+ –His 6 bridge [42,43]. These findings paved the way for reversible PEG-probe molecule linking on AFM tips via NTA·Ni 2+ –His 6 bonds (see Fig. 4), in other words to use the “metal chelating microscopy tip as a new toolbox for single molecule experiments by AFM” [43]. The first successful example of tip-PEG-probe link- ing via the NTA·Ni 2+ –His 6 bond is shown in Fig. 4. The NH 2 -functionalized tip was derivatized with mercaptopropionyl groups to which PDP–PEG–NTA was coupled via disulfide linkage. In the presence of Ni 2+ , His 6 -tagged MBP-VLDLR1-3 was tightly bound to the NTA·Ni 2+ function. Human VLDLR serves as a receptor for human rhinovirus serotype 2 (HRV2) and from binding studies using different recombinant soluble fragments of VLDLR the bind- ing site has been localized to the ligand binding repeats 2 and 3 [33,35]. Fortunately, Ni 2+ was also helpful to immobilize the virus particles on ultraflat mica by simple electrostatic adsorption. The viruses

Fig. 4. Flexible tethering of a His 6 -tagged probe molecule to an AFM tip. (A) The amino groups on the tip (see Fig. 1) were derivatized with mercaptopropionyl residues by reaction with SATP in chloroform/TEA, followed by cleavage of the S-acetyl groups with hydroxylamine. The resulting free thiol on the AFM tip was then reacted with the pyridyldithio group of PDP–PEG–NTA, yielding a stable disulfide bond. The NTA function was charged with Ni 2+ and an ectodomain fragment of the human VLDLR was specifically bound via its His 6 -tag. MBP-VLDLR1-3 contained the three N-terminal ligand binding repeats of human VLDLR fused

at the N-terminus to maltose-binding protein and at the C-terminus

to a hexa-His-tag. (B) Human rhinovirus particles (HRV2) were

electrostatically bound to freshly cleaved muscovite mica in 5 mM

Ni 2+ , yielding regular arrays of viruses. The AFM image shows

a surface of 130 m × 130 m.

were deposited in regular arrays, as can be seen in Fig. 4. The specific recognition of tip-linked MBP- VLDLR1-3 by the virus protein was measured in force–distance cycles as shown in Fig. 5A. In the ap- proach phase (dotted trace) the cantilever with the tip was lowered towards the substrate at a linear rate of

. In the ap- proach phase (dotted trace) the cantilever with the tip was lowered towards

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Riener et al. / Analytica Chimica Acta 497 (2003) 101–114 Fig. 5. The unbinding force of

Fig. 5. The unbinding force of the specific interaction between mica-bound human rhinovirus 2 and a single tip-linked VLDLR1-3 molecule (compare Fig. 4). The distance between the AFM tip

and the mica substrate (abscissa in panels A and B) was scanned

at 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 constant

of 35 pN nm 1 the linear scan rate of 60 nm s 1 corresponded to a

linear force velocity of 2.1 nN s 1 . Panel A was recorded in buffer

B which contained 0.1 mM Ni 2+ and 1 mM Ca 2+ in addition to

NaCl and Tris–HCl. The negative deflection in the retrace (solid trace in panel A) corresponded to a specific unbinding event with

38 pN rupture force. Panel B was recorded after 30 min incubation

in buffer C which contained 100 mM EGTA besides NaCl and

Tris–Cl. Panel C shows the probability density function (pdf)

of the unbinding forces measured in 40 retraces with a specific

unbinding event as exemplified in panel A. The most probable unbinding force (peak maximum) was 42 ± 4 pN.

60 nm s 1 . At the distance d = 0 nm, the apex of the tip made contact with the hard mica substrate. Further lowering of the tip led to an upward deflection of the cantilever, expressed in terms of bending force (y-axis in Fig. 5A) on the basis of the known spring constant of the cantilever (35 pN nm 1 ). Upon retraction of the cantilever holder (solid trace in Fig. 5A), the bending of the cantilever is relaxed until d = 0 nm is reached again. No force is observed during the next 10 nm of retraction but then a progressive downward deflec- tion is seen which reflects the specific adhesion of a single MBP-VLDLR1-3 molecule to a virus particle. The slope of the downward deflection is flatter than the slope at d < 0 nm, due to the higher elasticity of the PEG chain as compared to the cantilever itself. At d 16 nm, the specific adhesion is ruptured and the cantilever 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 unbinding events is shown in Fig. 5C. The peak maximum was found at an unbinding force of 42 ± 4 pN. The specificity of the interaction was demonstrated by its sensitivity to EGTA. After addition of EGTA, no specific adhesion events were seen in subsequent force–distance cycles, as exemplified by Fig. 5B. EGTA was expected to abolish specific binding in two ways. On the one hand, Ca 2+ ions are essential for the structural integrity of MBP-VLDLR1-3 [35]. On the other hand, Ni 2+ ions are required for main- taining the NTA·Ni 2+ –His 6 bridge between tip and MBP-VLDLR1-3. While it could not be discrimi- nated which divalent cation was first removed on the time scale of the AFM experiment, the EGTA block (as in Fig. 5B) indicated that the specific unbinding events (as in Fig. 5A) were caused by interaction of MBP-VLDLR1-3 with the virus particles. A unique feature of virus recognition by tip-tethered MBP-VLDLR1-3 is the relaxed cantilever position (zero force) at 0 d 10 nm in the solid trace of Fig. 5A. This must be attributed to the interplay between the height of the virus itself, the length of the PEG 800 crosslinker, and the position of PEG 800 attachment on the AFM tip. There are 60 symmetry-related receptor attachment sites distributed over the icosahedral virus surface, thus, binding sites on the side and on the top of the particle can be reached by those PEG–VLDLR tentacles also which are

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anchored higher up on the side of the AFM tip. This explains the nearly 10 nm tip–substrate distance at the begin of the downward deflection (see solid trace in Fig. 5A). Shorter and longer distances were also observed in other force–distance cycles but the 10 nm distance shown in Fig. 5A was most frequent. Fortunately, the NTA·Ni 2+ –His 6 linkage between

MBP-VLDLR1-3 and the PEG tentacles on the tip was quite stable; only after 1–2 h, the chance to see a specific recognition event in one force–distance cycle became noticeably lower, indicating a surprisingly slow dissociation of His 6 -tagged probe molecules from the tip. From this follows that His 6 -tagged probe molecules to be used in recognition force mi- croscopy/spectroscopy can reliably be tethered to AFM tips with PEG–NTA tentacles by the procedure shown in Fig. 4. It should be noted that the unbinding experiments shown in Fig. 5 were done at various loading rates (force increase per second), yielding a linear de- pendence of unbinding force upon the logarithm of loading rate (data not shown) which is typical for

a single energy barrier [44,45]. Moreover, the same

method was successfully used in our lab to recon- stitute the biospecific interaction between the small GTPase Ran and the nuclear import receptor importin 1 in the AFM, using PDP–PEG–NTA to tether His 6 -tagged Ran to the AFM tip (compare Fig. 4), while importin 1 was linked to functionalized mica via short (2 nm) spacers [46]. Here, again, the linear dependence of unbinding force upon loading rate was observed, and the unbinding force at a given loading rate was switched from high to low when bound GTP was hydrolyzed to GDP by the small GTPase Ran. Such large, consistent data sets can justly be taken as verification of the coupling scheme shown in Fig. 4 even though our usual macroscopic marker enzyme assay of coupling site density [13,23] could not be done, due to lack of a His 6 -tagged marker enzyme.

3.4. Comparison of covalent versus noncovalent tethering of probe molecules to AFM tips

Covalent linking of probe molecules to AFM tips

is very straightforward when the probe molecule al-

ready contains a free sulfhydryl (compare Fig. 1), as is the case with a number of native proteins, with genetically engineered cysteine mutants, or with prop-

erly synthesized peptides, oligonucleotides or other small molecules. In any case, special care must be taken to prevent sulfhydryl oxidation since protective agents like DTT or 2-mercaptoethanol cannot be used for obvious reasons. Proteins without free sulfhydryl groups, however, need to be pre-derivatized with SATP to introduce S-acetyl-protected mercaptopropi- onyl groups which can be coupled to PDP groups after de-acetylation with hydroxylamine (see Fig. 1) [8,24]. The pre-derivatization requires at least 0.1–0.2 mg of protein, preferably at 1 mg ml 1 protein concen- tration. Free SATP must rigorously be removed by gel filtration which causes sample dilution, and the aliquots of the diluted protein may denature upon refreezing. No such limitations are experienced when His 6 - tagged proteins are tethered to AFM tips. His 6 is a most popular tag allowing for purification of an over- expressed protein in a single step on an NTA–Ni 2+ column. The same tag now provides for a long-lasting link to PEG–NTA–Ni 2+ tentacles on AFM tips, as depicted in Fig. 4, thereby eliminating the need for chemical pre-derivatization of the probe molecule. Af- ter purification, the His 6 -protein can immediately be frozen in small aliquots and only one aliquot at a time needs to be thawed and incubated with the function- alized AFM tip. Due to the stability of the His 6 -tag, even reuse of the same protein aliquot should be pos- sible in case of proper storage conditions, whereas the free thiol needed for covalent coupling (Fig. 1) is sensitive to oxidation. As a consequence, noncovalent coupling (Fig. 4) is the method of choice if the probe molecule is already available as a His 6 -tagged protein. The same applies when the protein has been cloned and extensive recog- nition force studies are intended. In contrast, covalent coupling (Fig. 1) is much more appropriate for short term studies with untagged proteins or other probe molecules. Thus, the two methods nicely complement each other, and the tools for both methods are now available, 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

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

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