Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy

Martina Rangl, Michael Leitner, Tiina Riihimäki, Soili Lehtonen, Vesa P. Hytönen, Hermann J. Gruber, Markku Kulomaa, Peter Hinterdorfer, Andreas Ebner

Research output: Contribution to journalArticleScientificpeer-review

4 Citations (Scopus)


Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (koff) and energy landscape distances (xβ) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the xβ values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications.

Original languageEnglish
Pages (from-to)92-97
Number of pages6
JournalJournal of Molecular Recognition
Issue number2
Publication statusPublished - 2014
Publication typeA1 Journal article-refereed


  • atomic force microscopy
  • avidin
  • molecular recognition force spectroscopy
  • protein recognition
  • single molecule
  • testosterone

Publication forum classification

  • Publication forum level 1


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