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Biophysics Researchers: Constant Calcium Uptake Causes Cancer in Cells

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[2017/Nov/28] Dr. Irene Frischauf and Dr. Rainer Schindl made the cover of Science Signaling with their publication: Calcium regulates many vitally important functions in the body - if the calcium balance is defective, diseases like cancer develop... ...  more of Biophysics Researchers: Constant Calcium Uptake Causes Cancer in Cells (Titel)

Noteworthy Publication in Angewandte Chemie

[2016/Nov/06] Publication in Angewandte Chemie: "Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins" ...  more of Noteworthy Publication in Angewandte Chemie (Titel)

Victoria Lunz - Genuinely Passionate about Research

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Tuning membrane protein mobility by confinement into nanodomains

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[2016/11/16] In cooperation with Prof. Peter Pohl (Institute of Biophysics, JKU), lead CBL researchers DI Andreas Karner and Dr. Johannes Preiner have developed a platform to study membrane proteins. ...  more of Tuning membrane protein mobility by confinement into nanodomains (Titel)

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Functionalization of AFM tips and flat supports for recognition force microscopy

Atomic force microscopy (AFM) can operate in aqueous solution under physiological conditions and reveal fine details not resolved by electron microscopy. AFM is thus well suited for structural analysis of biomolecules and their assemblies. In addition, the AFM tip can be upgraded into a monomolecular biosensor by coupling of a "ligand" molecule which is moved over the sample surface and recognized by complementary "receptor" molecules on the sample surface, yielding a map of recognition sites [31-33]. Binding is detected as a rupture event which is sensed by a vertically oscillated cantilever, preferably under simultaneous monitoring of sample topography [31-33]. Moreover, fine biophysical and structural details of biomolecular interactions are obtained when measuring the unbinding forces at different force loading rates [17, 19], interaction timse before dissociation [30], biochemical modifications and co-association of third components [17], or with different mutants or truncated forms of the participating proteins [6].
Linear polyethylene glycol (PEG) chains have frequently been used to attach one or few sensor molecules to an AFM tip because PEG is chemically and physically inert and it allows for rapid and free reorientation of the sensor molecule when the AFM tip approaches the surface [1-17, 19-28, 30-38]. Thus, even a single sensor molecule on the tip can recognize its cognate molecule on the target surface with up to 25% probability in one force-distance cycle with 100-800 nm vertical range and 1 s cycle time [2]. Moreover, the non-linear stretching behavior of the PEG tether helps to discriminate biospecific interactions from nonspecific tip-sample adhesion [2, 5, 12].
Heterobifunctional PEG linkers with a length of ~6 nm are used to tether proteins to AFM tips, as shown in Fig. 1A and Fig. 1B. Both methods require tips with amino groups. These are introduced by one of the methods shown in Fig. 2. The NH2-function on the tip is reacted with the NHS ester of the PEG linker, resulting in unilateral attachment of PEG by stable amide bond. In the next step, the protein is attached to the free-tangling end of the PEG chain, either via disulfide bond formation (Fig. 1A) or via amine-aldehyde linkage (Fig. 1B). The latter method has the advantage that no free thiol function (SH) is required on the protein of interest. This is particularly important in the case of extracellular proteins, such as antibodies, which usually lack free cysteine residues. The aldehyde method (Fig. 1B) was first used in 2005 [6, 19, 37], before this time it was necessary to pre-derivatize antibodies with SATP (as depicted Fig. 1A).


Each of the two coupling schemes in Fig. 1 has been characterized on a quantitative level [5, 6]. For this purpose, the antibodies were “decorated” with biotin residues (symbolized by little blue spheres in Fig. 1). Biotin residues were chosen (i) because they have a very high affinity for a tetrameric protein called avidin, and (ii) because a monolayer of avidin can be formed on freshly cleaved mica within minutes by simple adsorption (see Fig. 1). In the AFM, the tip is repeatedly brought in contact with the surface, and specific binding of biotin to avidin leads to characteristic rupture events when the tip is retracted from the sample surface [2, 5, 6].
Fig. 2A depicts three reagents which can be chosen to introduce amino groups on silicon nitride tips. Two criteria must be met: The tip must not get sticky and the amino group density should be relatively low so that only few PEG linkers are attached at the apex of the tip. In our two studies on aminofunctionalization we learned that the choice of reagent is less important than the exact reaction conditions [2, 25]. In these studies, the NH2-groups on the silicon nitride surface were derivatized with biotin-PEG-NHS, resulting in surface-linked PEG tentacles with biotin end groups (blue sphere in Fig. 2B). The surface density of these biotin functions was judged from the number of avidin-peroxidase molecules that was specifically bound to the surface [2, 25]. Nonspecific binding was measured by blocking all avidin molecules with free biotin.

The following probe molecules have been tethered to the AFM tip:

  • Antibodies [1, 4, 5, 6, 9, 10, 16, 22, 31, 32, 37, 38]
  • Cadherin [30, 35]
  • Cytochrome C551 [19]
  • Biotin [2, 33, 34]
  • Thioglucose [22, 24, 28]
  • Phlorizin [16]
  • His6-proteins or peptides [11, 12, 17, 29]

Immobilization of target molecules on ultra-flat supports for AFM studies:

  • Adsorption to mica [2, 3, 5, 6, 10, 31, 33]
  • Aminofunctionalized mica [1, 4, 9, 17, 30, 32]
  • Aminofunctionalized silicon nitride [25]
  • Covalent binding to glass [18]
  • Ultra-flat gold [11, 12, 19, 29, 35, 46]

Core papers on tip and support functionalization

  1. Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K., and Schindler*, H. (1996) Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc. Natl. Acad. Sci. USA 93, 3477-3481.
  2. Riener, C. K., Stroh, C. M., Ebner, A., Klampfl, C., Gall, A. A., Romanin, C., Lyubchenko, Y. L., Hinterdorfer, P., and Gruber*, H. J. (2003) Simple Test System for Single Molecule Recognition Force Microscopy. Anal. Chim. Acta 479, 59-75.
  3. Riener, C. K., Kienberger, F., Hahn, C. D., Buchinger, G. M., Egwim, I. O. C., Haselgrübler, T., Ebner, A., Romanin, C., Klampfl, C., Lackner, B., Prinz, H., Blaas, D., Hinterdorfer, P., and Gruber*, H. J. (2003) Heterobifuctional crosslinkers for linking of single ligand molecules to scanning probes. Anal. Chim. Acta 497, 101-114.
  4. Klein, D. C. G., Stroh, C. M., Jensenius, H., van Es, M., Kamruzzahan, A. S. M., Stamouli, A. , Gruber, H. J., Oosterkamp, T. H., and Hinterdorfer, P.* (2003) Covalent Immobilization of Single Proteins on Mica for Molecular Recognition Force Microscopy. ChemPhysChem. 4, 1367-1371.
  5. Kamruzzahan, A. S. M., Ebner, A., Wildling, L., Kienberger, F., Riener, C. K., Hahn, C. D., Pollheimer, P. D., Winklehner, P., Hölzl, M., Lackner, B., Schörkl, D. M., Hinterdorfer, P., and Gruber, H. J. (2006) Antibody linking to atomic force microscope tips via disulfide bond formation. Bioconjugate Chem. 17, 1473-1481.
  6. Ebner, A., Wildling, L., Kamruzzahan, A. S. M., Rankl, C., Wruss, J., Hahn, C. D., Hölzl, M., Kienberger, F., Blaas, D., Hinterdorfer, P., and Gruber, H. J.* (2007) A new, simple method for linking of antibodies to atomic force microscopy tips. Bioconjugate Chem. 18, 1176-1184.
  7. Ebner, A., Wildling, L., Zhu, R., Haselgrübler, Th., Hinterdorfer, P., and Gruber, H. J.* (2007) Functionalization of Probe Tips and Supports for Single Molecule Recognition Force Microscopy. Top. Curr. Chem., invited review, in press.

Further studies with new methodical details on tip/support functionalization:

8. Haselgrübler, T., Amerstorfer, A., Schindler, H., and Gruber*, H. J. (1995) Synthesis and applications of a new poly(ethylene glycol) derivative for the crosslinking of amines with thiols. Bioconjugate Chem. 6, 242-248.

9. Hinterdorfer*, P., Schilcher, K., Baumgartner, W., Gruber, H. J., and Schindler, H. (1998) A Mechanistic Study of the Dissociation of Individual Antibody-Antigen Pairs by Atomic Force Microscopy. Nanobiology 4, 177-188.

10. Willemsen, O. H., Snel, M. M. E., van der Werf, K. O., Gruber, H. J., Hinterdorfer, P., Schindler, H., van Kooyk, Y., de Grooth, B.G., Greve, J., and Figdor*, C.G. (1998) Simultaneous Height and Adhesion of Antibody-Antigen Interactions by Atomic Force Microscopy. Biophys. J. 75, 2220-2228.

11. Kienberger, F., Kada, G., Gruber, H. J., Pastushenko, P., Riener, C., Trieb, M., Knaus, H.-G., Schindler, H., and Hinterdorfer*, P. (2000) Recognition Force Spectroscopy Studies of the NTA-His6 Bond. Single Molecules 1, 25-31.

12. Kienberger, F., Pastushenko, V. P., Kada, G., Gruber, H. J., Riener, C., Schindler, H., and Hinterdorfer*, P. (2000) Static and Dynamical Properties of Single Poly(Ethylene Glycol) Molecules Investigated by Force Spectroscopy. Single Molecules 1, 123-128.
13. Hinterdorfer*, P., Kienberger, F., Raab, A., Gruber, H. J., Baumgartner, W., Kada, G., Riener, C., Wielert-Badt, S., Borken, C., and Schindler, H. (2000) Poly(Ethylene Glycol): An Ideal Spacer for Molecular Recognition Force Microscopy/Spectroscopy. Single Molecules 1, 99-103.

14. Hinterdorfer, P., Gruber, H. J., Kienberger, F., Kada, G., Riener, C., Borken, C., and Schindler, H. (2002) Surface attachment of ligands and receptors for molecular recognition Force microscopy. Coll. Surf. B 23, 115-123.
(Colloids and Surfaces B: Biointerfaces)

15. Riener, C. K., Kada, G., Borken, C., Kienberger, F., Hinterdorfer, P., Schindler, H., Schütz, G. J., Schmidt, T., Hahn, C. D., and Gruber*, H. J. (2002) Bioconjugation for biospecific detection of single molecules in atomic force microscopy (AFM) and in single dye tracing (SDT). Recent Research Developments in Bioconjugate Chemistry 1, 133-149.

16. Wielert-Badt, S., Hinterdorfer*, P., Gruber, H. J., Lin, J.-T., Badt, D., Wimmer, B., Schindler, H., and Kinne, R. K.-H. (2002) Single molecule recognition of protein binding epitopes in brush border membranes by force microscopy. Biophys. J. 82, 2767-2774.

17. Nevo, R., Stroh, C. M., Kienberger, F., Kaftan, D. Brumfeld, V., Elbaum, M., Reich, Z., and Hinterdorfer, P.* (2003) A molecular switch between alternative conformational states in the complex of Ran and importin 1. Nat. Struct. Biol. 10, 553-557.

18. Ebner, A., Kienberger, F., Stroh, C. M., Gruber, H. J., and Hinterdorfer, P.* (2004) Monitoring of Glass Derivatization with Pulsed Force Mode Atomic Force Microscopy. Micros. Res. Tech 65, 246-251.

19. Bonanni, B., Kamruzzahan, A. S. M., Bizzarri, A. R., Rankl, C., Gruber, H. J., Hinterdorfer, P., and Cannistraro, S.* (2005) Single molecule recognition between cytochrome C 551 and gold-immobilized azurin by force spectroscopy. Biophys.l J. 89, 2783-2791.

20. Kienberger, F., Ebner, A., Gruber, H. J., and Hinterdorfer, P.* (2006) Molecular Recognition Imaging and Force Spectroscopy of Single Biomolecules. Accounts Chem. Res. 39, 29-36.

21. Kienberger,* F., Gruber, H., and Hinterdorfer, P. (2006) Dynamic force microscopy and spectroscopy. Applied scanning probe methods. Volume 2: Scanning probe microscopy techniques (Bushan, B., and Fuchs, H., Eds.) pp 143-164, Chapter 5, Springer-Verlag, Berlin.

22. Puntheeranurak, T., Wildling, L., Gruber, H. J., Kinne, R. K., and Hinterdorfer, P.* (2006) Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells. J. Cell. Sci. 114, 2960-2967.

23. Ebner, A., Madl, J., Kienberger, F., Chtcheglova, L. A., Puntheeranurak, T., Zhu, R., Tang, J., Stroh, C., Gruber, H. J., Schütz, G. J., and Hinterdorfer, P.* (2007) Single molecule force microscopy on cells and biological membranes. Curr. Nanosci. 3, 49-56.

24. Puntheeranurak, T., Wimmer, B., Castaneda, F., Gruber, H. J., Hinterdorfer, P., Kinne, R. K. H.* (2007) Substrate Specificity of Sugar Transport by Rabbit SGLT1: Single molecule AFM versus Transport Studies. Biochemistry, 47, 2797-2804.

25. Ebner,* A., Hinterdorfer, P., and Gruber, H. J. (2007) Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers. Ultramicroscopy 107, 922-927.

26. Kienberger, F., Ebner, A., Chtcheglova, L., Wildling, L., Puntheeranurak, T., Gruber, H. J., and Hinterdorfer, P. (2007) AFM-Tip Chemistry for Single Molecule Recognition Imaging. NSTI Nanotech. 2, 557-560.

27. Kienberger,* F., Chtcheglova, L., Ebner, A., Puntheeranurak, T., Gruber, H., and Hinterdorfer, P. (2007) Single-molecule studies on cells and membranes using the atomic force microscope. Applied scanning probe methods. Volume 6: (Bushan, B., and Fuchs, H., Eds.) pp 101-125, Chapter 14, Springer-Verlag, Berlin.

28. Puntheeranurak T., Kinne R.K.H., Gruber H.J., and Hinterdorfer P. (2007) Single-Molecule AFM Studies of Substrate Transportation of Sodium-Glucose Cotransporter SGLT1. Journal of the Korean Physical Society, in press.

29. Verbelen, C., Gruber, H. J. and Dufrêne, Y. F. (2007) The NTA-His6 bond is strong enough for AFM single-molecular recognition studies. J. Mol. Rec., in press.

Further AFM studies on biospecific recognition with functionalized tips

30. Baumgartner, W., Gruber, H., Hinterdorfer, P., and Drenckhahn*, D. (2000) Affinity of Trans-Interacting VE-Cadherin Determined by Atomic Force Microscopy. Single Molecules 1, 123-128.

31. Stroh, C. M., Ebner, A., Geretschläger, M., Freudenthaler, G., Kienberger, F., Kamruzzahan, A. S. M., Smith-Gill, S. J., Gruber, H. J., and Hinterdorfer, P.* (2004) Simultaneous Topography and Recognition Imaging Using Force Microscopy. Biophys. J. 87 (3), 1981-1990.

32. Stroh, C. M., Wang, H., Bash, R., Ashcroft, B., Nelson, J., Gruber, H., Lohr, D., Lindsay, S. M.*, and Hinterdorfer, P. (2004) Single-molecule recognition imaging microscopy. Proc. Natl. Acad. Sci. USA, 101, 12503-12507.

33. Ebner, A., Kienberger, F., Kada, G., Stroh, C. M., Geretschläger, M., Kamruzzahan, A. S. M., Wildling, L., Johnson, W. T., Ashcroft, B., Nelson, J., Lindsay, S. M., Gruber, H. J., and Hinterdorfer, P.* (2005) Localization of single avidin-biotin interactions using simultaneous topography and molecular recognition imaging. ChemPhysChem. 6, 897-900.

34. Ebner, A., Kienberger, F., Huber, C., Kamruzzahan, A. S., Pastushenko, V. P., Tang, J., Kada, G., Gruber, H. J., Sleytr, U. B., Sara, M., and Hinterdorfer, P.* (2006) Atomic-force-microscopy imaging and molecular-recognition-force microscopy of recrystallized heterotetramers comprising an S-layer-streptavidin fusion protein. ChemBioChem. 7, 588-591.

35. Waschke, J., Menendez-Castro, C., Bruggeman, P., Koob, R., Amagai, M.; Gruber, H. J., Drenckhahn, D., and Baumgartner, W.* (2007) Imaging and Force Spectroscopy on Desmoglein 1 Using Atomic Force Microscopy Reveal Multivalent Ca2+-Dependent, Low-Affinity Trans-Interaction. J. Membrane Biol. 216, 83-92.

36. Tang, J., Krajcikova, D., Zhu, R., Ebner, A., Gruber, H. J., Barak, I., and Hinterdorfer, P.* (2007) Atomic Force Microscopy Imaging and Molecular Recognition Force Spectroscopy of Coat Proteins on the Surface of Bacillus subtilis Spore. J. Mol. Rec., in press.

Patent applications involving functionalized AFM tips:

37. Gruber, H. J., and Johnson, W. T. (2006) Probe and method of making a probe using crosslinker compositions as chemical and biological sensors. WO/2007/084238.

38. Zhu, R., Hinterdorfer, P., and Gruber, H. (2007) Method for detecting 5-methylcytosine. WO/2007/087653.