Heterobifunctional crosslinkers for tethering single ligand molecules to scanning probes

• Published in: Analytica chimica acta Vol. 497; no. 1; pp. 101 - 114
• Main Authors: Riener, Christian K., Kienberger, Ferry, Hahn, Christoph D., Buchinger, Gerhard M., Egwim, Innocent O.C., Haselgrübler, Thomas, Ebner, Andreas, Romanin, Christoph, Klampfl, Christian, Lackner, Bernd, Prinz, Heino, Blaas, Dieter, Hinterdorfer, Peter, Gruber, Hermann J.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-11-2003; Elsevier
• Subjects: Atomic force microscope; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Heterobifunctional crosslinker; Human rhinovirus; Interactions. Associations; Intermolecular phenomena; Molecular biophysics; Poly(ethylene glycol); Recognition force spectroscopy; DIEA; RT; DTT; HRV2; AFM; MBP-VLDLR1-3; SMRFM; VLDLR; lys-DA; NH 2–PEG–NH 2; PDP-OH; Boc 2O; Human rhinovirus; SPDP; X–PEG–NH 2; BSA; Heterobifunctional crosslinker; X–PEG–X; Boc; Poly(ethylene glycol); NHS; NH 2–PEG–COOH; DMF; PDP–PEG–NHS; PDP; LC-SMCC; biotin–PEG–NHS; DCC; ESI-MS; mal–PEG–NHS; SATP; EGTA; Tris; Recognition force spectroscopy; glu; TEA; NMR; PEG; Atomic force microscope; NTA; PDP–PEG–NTA; VS–PEG–NHS; Atomic force microscopy; Molecular association; Molecular recognition; Molecular interaction; Molecular complex; Biological macromolecule; Experimental study
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2003.08.041

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 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 β1 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].