Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Self-assembly of fungal proteins (#207)

Mahiar Mahjoub 1 , Georg Meisl 2 , Ruihao Li 1 , Chi Le Lan Pham 3 , Sam Walker 1 , Margie Sunde 3 , Ann Kwan 1
  1. School of Life and Environmental Science, University of Sydney, Sydney, NSW, Australia
  2. Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
  3. Discipline of Pharmacology, School of Medical Science, University of Sydney, Sydney, NSW, Australia

Class I hydrophobins are fungal proteins that are able to spontaneously self-assemble to form robust amphipathic amyloid fibrils, known as rodlets, upon encountering hydrophobic:hydrophilic interfaces. Hydrophobins are characterised with eight conserved cysteine residues forming four disulphide bonds. In EAS, a class I hydrophobin from Neurospora crassa, two regions of interest are known: (1) the flexible Cys3-4 loop (C19-C45) with unknown function and (2) the Cys7-8 loop (C61-C80) implicated as the region responsible for amyloidogenesis. Furthermore, despite the presence of a large exposed hydrophobic surface, which is located away from the charged residues, the protein remains highly soluble. Thus, this project aims to elucidate the role of Cys3-4 loop by comparing the rodlet assembly of EAS, and its engineered variant EASΔ15 which contains a substantially shortened and more structured Cys3-4 loop. In addition, the detection of intermediary hydrophobins transitioning from the soluble to the rodlet state can guide the elucidation of molecular mechanisms underlying rodlet assembly.

In this poster, several findings will be presented. Firstly, the concentration-dependent inhibition effect observed for EAS assembly implicates Cys3-4 as the main agent responsible for the inhibitory effect at higher concentrations. This discovery lays the ground-work for further biophysical investigations of the mechanisms of inhibition by the Cys3-4 loop using Nuclear Magnetic Resonance (NMR) spectroscopy. Secondly, the assembly kinetics data for both EAS and EASΔ15 were used for the selection and specialisation of generalised aggregation mathematical models so they can be also used to describe hydrophobin assembly with its unique interfacial requirements. Thirdly, mutational studies of charged residues in EAS highlighted their role in impeding or boosting rodlet assembly. Lastly, preliminary NMR exchange experiments have so far been unsuccessful in the detection of intermediary protein species (e.g. with a conformationally changed Cys7-8 loop) during rodlet formation, warranting the need for further optimisation.

  1. Kwan, A. H., Winefield, R. D., Sunde, M., Matthews, J. M., Haverkamp, R. G., Templeton, M. D., and Mackay, J. P. (2006) Structural basis for rodlet assembly in fungal hydrophobins. Proc Natl Acad Sci USA 103, 3621-3626