Molecular interactions of hydrophobin proteins with their surroundings

Aalto University, P.O. Box 11000, FI-00076 Aalto www.aalto. fi Author Mathias S. Grunér Name of the doctoral dissertation Molecular interactions of hydrophobin proteins with their surroundings Publisher School of Chemical Technology Unit Department of Biotechnology and Chemical Technology Series Aalto University publication series DOCTORAL DISSERTATIONS 206/2015 Field of research Biotechnology Manuscript submitted 5 June 2015 Date of the defence 10 December 2015 Permission to publish granted (date) 6 November 2015 Language English Monograph Article dissertation (summary + original articles) Abstract This thesis describes the properties of a group of proteins named hydro-phobins, which fulfil a variety of functions in the growth and function of filamentous fungi. Hydrophobins can be utilized as coatings/protective agents, in adhesion, in surface modifications and overall functions that require surfactant-like properties. This work is concentrated on the hydrophobins HFBI, HFBII and HFBIII expressed by Trichoderma reesei. The aims of this study were to examine in what manner hydrophobins function when interacting with their surroundings and how their surroundings affect their function. Hydrophobins were shown strongly to adhere to surfaces of varying polarity and structure by self-assembly, governed by their amphiphilic nature, and to adsorb with different orientation on hydrophilic and hydrophobic surfaces. The proteins were shown to selectively recruit other proteins and molecules to a self-assembled amphiphilic film of hydrophobin. HFBI variants bound to a surface were shown to recruit T. reesei enzymes specifically depending on localized protein surface charge on the hydrophilic part of the protein, and HFBII adsorbed on nanoparticles was shown to bind layers of human plasma proteins in different manner when adsorbed on nanoparticles of varying polarity. Surface films formed by hydrophobins were shown to be highly elastic, and charged residues on the side of the proteins were shown to have a role in stabilizing the protein films formed. The surroundings in which the proteins exist were shown to also affect their function. Surfaces of varying polarity in the protein surroundings affected how they self-assemble, and hydrophobin multimer exchange in solution was shown to be governed by hydrophobic interactions and the multimer exchange behaviour was shown to be affected by other proteins and molecules. HFBII and HFBI were shown to interact in solution, altering multimer kinetics and thermodynamics considerably. Solution association methods, surface characterization analysis methods and size measurement techniques such as stopped-flow spectroscopy, quartz crystal microbalance with dissipation and differential centrifugal sedimentation were used. The results presented here show that hydrophobins function by selectively interacting with their surroundings assembled at various interfaces specifically recruiting other proteins and molecules and that the surroundings in which the proteins exist also affects their function in terms of multimer exchange behaviour and surface adhesion properties. The knowledge learned here regarding hydrophobins, show that these proteins can be specialized to function as highly selective self-assembling building blocks in applications such as biosensors and biocompatible coatings, and gives new insight in the growth and function of filamentous fungi.