Quantifying biomolecular hydrophobicity: Single molecule force spectroscopy of class II hydrophobins

Arja Paananen (Corresponding Author), Sabine Weich, Géza R. Szilvay, Michael Leitner, Kirsi Tappura, Andreas Ebner (Corresponding Author)

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Hydrophobins are surface-active proteins produced by filamentous fungi. The amphiphilic structure of hydrophobins is very compact, containing a distinct hydrophobic patch on one side of the molecule, locked by four intramolecular disulphide bridges. Hydrophobins form dimers and multimers in solution to shield these hydrophobic patches from water exposure. Multimer formation in solution is dynamic, and hydrophobin monomers can be exchanged between multimers. Unlike class I hydrophobins, class II hydrophobins assemble into highly ordered films at the air-water interface. In order to increase our understanding of the strength and nature of the interaction between hydrophobins, we used atomic force microscopy (AFM) for single molecule force spectroscopy to explore the molecular interaction forces between class II hydrophobins from Trichoderma reesei under different environmental conditions. A genetically engineered hydrophobin variant, NCys-HFBI, enabled covalent attachment of proteins to the apex of the AFM cantilever tip and sample surfaces in controlled orientation with sufficient freedom of movement to measure molecular forces between hydrophobic patches. The measured rupture force between two assembled hydrophobins was ∼31 pN, at a loading rate of 500 pN/s. The results indicated stronger interaction between hydrophobins and hydrophobic surfaces than between two assembling hydrophobin molecules. Furthermore, this interaction was stable under different environmental conditions, which demonstrates the dominance of hydrophobicity in hydrophobin-hydrophobin interactions. This is the first time that interaction forces between hydrophobin molecules, and also between naturally occurring hydrophobic surfaces, have been measured directly at a single-molecule level.
Original languageEnglish
Article number100728
Number of pages10
JournalJournal of Biological Chemistry
Publication statusPublished - 3 May 2021
MoE publication typeA1 Journal article-refereed


This work was supported by the Academy of Finland (Grant #126572) and by the Linz Institute of Technology (LIT) - project LIT-2019-7-SEE-113, the government of Upper Austria, and the Federal Ministry of Education, Science and Research (BMBWF). The work was carried out under the Academy of Finland’s Centers of Excellence Programme (2014–2019).


  • single molecule force spectroscopy
  • SMFS
  • hydrophobic interaction
  • hydrophobin
  • HFBI
  • Trichoderma reesei
  • class II hydrophobin


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