European Biophysics Journal

, Volume 35, Issue 1, pp 72–78 | Cite as

Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy

  • Stefan K. Kufer
  • Hendrik Dietz
  • Christian Albrecht
  • Kerstin Blank
  • Angelika Kardinal
  • Matthias Rief
  • Hermann E. Gaub
Article

Abstract

A genetically modified form of the human DNA repair protein O6-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.

Keywords

Molecular recognition  SPR  AFM  Suicide coupler  hAGT  SNAP-tag 

Abbreviations

hAGT

O6-alkylguanine-DNA-alkyltransferase

GST

Glutathione S-Transferase

PEG

Polyethylene glycol

BG

Benzylguanine

SPR

Surface plasmon resonance

AFM

Atomic force microscope

EDC

1-ethyl-3-(3-diaminopropyl)carbodiimide hydrochloride

NHS

N-hydroxysuccinimide

GST

Glutathione S-transferase

CMC

Carboxymethylcellulose

References

  1. Butt HJ, Jaschke M (1995) Calculation of thermal noise in atomic force microscopy. Nanotechnology 6:1–7CrossRefADSGoogle Scholar
  2. Damoiseaux R, Keppler A, Johnsson K (2001) Synthesis and applications of chemical probes for human O6-alkylguanine-DNA alkyltransferase. Chembiochem 2:285–287CrossRefPubMedGoogle Scholar
  3. Daniels DS, Tainer JA (2000) Conserved structural motifs governing the stoichiometric repair of alkylated DNA by O(6)-alkylguanine-DNA alkyltransferase. Mutat Res 460:151–163PubMedGoogle Scholar
  4. Daniels DS, Mol CD, Arvai AS, Kanugula S, Pegg AE, Tainer JA (2000) Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. Embo J 19:1719–1730PubMedCrossRefGoogle Scholar
  5. Dietz H, Rief M (2004a) Single molecule force spectroscopy of proteins. In: Proceedings of NATO ASI and SUSSP 59th. Soft condens Matter Phys Mol cell Biol (in press)Google Scholar
  6. Dietz H, Rief M (2004b) Exploring the energy landscape of green fluorescent protein by single molecule mechanical experiments. PNAS 101:16192–16197CrossRefPubMedADSGoogle Scholar
  7. Florin E-L, Rief M, Lehmann M, Ludwig M, Dornmair C, Moy T, Gaub HE (1995) Sensing specific molecular interactions with the atomic force microscope. Biosensors Bioelectronics 10:895–901CrossRefGoogle Scholar
  8. Gendreizig S, Kindermann M, Johnsson K (2003) Induced protein dimerization in vivo through covalent labeling. J Am Chem Soc 125:14970–14971CrossRefPubMedGoogle Scholar
  9. Grandbois M, Beyer M, Rief M, Clausen-Schaumann H, Gaub HE (1999) How strong is a covalent bond?. Science 283:1727–1730CrossRefPubMedADSGoogle Scholar
  10. Juillerat A, Gronemeyer T, Keppler A, Gendreizig S, Pick H, Vogel H, Johnsson K (2003) Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem Biol 10:313–317CrossRefPubMedGoogle Scholar
  11. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89CrossRefPubMedGoogle Scholar
  12. Kindermann M, George N, Johnsson N, Johnsson K (2003) Covalent and selective immobilization of fusion proteins. J Am Chem Soc 125:7810–7811CrossRefPubMedGoogle Scholar
  13. Neuert G, Kufer SK, Benoit M, Gaub HE (2004) Modular multichannel surface plasmon spectrometer. Review of Scientific Instruments (in press)Google Scholar
  14. Oesterhelt F, Rief M, Gaub HE (1999) Single molecule force spectroscopy by AFM indicates helical structure of poly(ethylene-glycol) in water. New J Phys, pp 6.1–6.11Google Scholar
  15. Pegg AE, Boosalis M, Samson L, Moschel RC, Byers TL, Swenn K, Dolan ME (1993) Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine. Biochemistry 32:11998–12006CrossRefPubMedGoogle Scholar
  16. Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276:1109–1112CrossRefPubMedGoogle Scholar

Copyright information

© EBSA 2005

Authors and Affiliations

  • Stefan K. Kufer
    • 1
  • Hendrik Dietz
    • 2
  • Christian Albrecht
    • 1
  • Kerstin Blank
    • 1
  • Angelika Kardinal
    • 1
  • Matthias Rief
    • 2
  • Hermann E. Gaub
    • 1
  1. 1.Lehrstuhl für Angewandte Physik, Sektion PhysikLudwig-Maximilians-Universität München and Center for NanoScienceMunichGermany
  2. 2.Physik-Department E22Technische Universität MünchenGarchingGermany

Personalised recommendations