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

    Year: 2021

    Journal: J. Biol. Chem., Volume 296, JAN-JUN

    Authors: Paananen, Arja; Weich, Sabine; Szilvay, Geza R.; Leitner, Michael; Tappura, Kirsi; Ebner, Andreas

    Organizations: Academy of Finland [126572]; Linz Institute of Technology (LIT) [LIT-2019-7-SEE-113]; government of Upper Austria; Federal Ministry of Education, Science and Research (BMBWF)

    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 disulfide 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 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 atomic force microscopy 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 similar to 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.
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