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

Arja Paananen,Sabine Weich,Géza R Szilvay,Michael Leitner,Kirsi Tappura,Andreas Ebner

doi:10.1016/j.jbc.2021.100728

Abstract

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 ∼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.

Highlights

Hydrophobins are fascinating proteins owing to their unique structure: instead of burying hydrophobic residues inside the protein structure, these proteins contain four disulfide bridges that stabilize a nanometer-sized hydrophobic patch on one side of the molecule
We wanted to gain an insight about the hydrophobicity and strength between two natural biomolecular hydrophobic surfaces
The later studies showed that observed long-range interactions seemed to depend greatly on the force-measuring techniques and methods for surface hydrophobization, and that the true hydrophobic interaction is short-ranged (

Summary

Introduction

By performing SMFS with the single class II hydrophobins tethered on the tip as well as the covalently immobilized ones on the surface, we could show a rupture force of $31 pN for the homophilic hydrophobin interaction at a loading rate of 500 pN/s. In the second specificity proof experiment performed in PBS, we added free hydrophobin molecules to the measurement configuration to allow complex formation both on the tip and on the surface to block the hydrophobic patches (see Fig. 3B).

Results

Conclusion