Synchrotron radiation circular dichroism spectroscopy of hydrophobins

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Plasmid design

Hydrophobins and their mutants were produced as fusion proteins with ubiquitin and an N-terminal hexahistidine tag. The sequence contains a cleavage site (either for TEV or Ubp41 proteases, (Pille et al., 2015) between ubiquitin and the hydrophobin (Fig. 22). Ubiquitin was used as a fusion protein to help maintain soluble the unfolded protein and intermediates during the in vitro oxidative folding step (see below). It was chosen for its small size and because it does not contain cysteines like GST, another protein often used to express and purify proteins. RodA and RodB were cloned in a pET15b plasmid (Novagen) coding for ampicillin resistance and a TEV cleavage site. A pET28b vector (Novagen) with kanamycin resistance coding for an Ubp41 cleavage site was used for RodC and RodC single point mutants. Multiple point mutants were cloned in a pET15b (ampicillin resistance and TEV cleavage site).

Expression and purification

Plasmids were transformed using heat shock in Escherichia coli. RodA and RodB were produced using the BL21 (DE3) strain. For RodC, we additionally tested Origami B (Novagen) cells in an attempt to produce native oxidized proteins in the cytoplasm. For Rod E and F, we also tried the Shuffle (New England Biolabs) strain. The Origami and Shuffle strains can in principle allow the production of disulfide containing proteins in the cytoplasm because both lack the thioredoxin and glutathione reductases. Additionally, the Shuffle strain contains the disulfide isomerase DsbC and cytoplasmic chaperones (DnaKJE, GroELS, ClpB) to facilitate the exchange of S-S and favor the formation of native S-S linkages (de Marco, 2009). For RodC, low yield of soluble and folded protein was obtained; RodC and its mutants were therefore produced as inclusion bodies using standard BL21 (DE3) E. coli strain. Importantly, WT RodC produced in the cytoplasm or oxidatively folded in vitro gave the same 1H -15N HSQC spectra.
The same expression and purification protocols were followed for all native and mutated hydrophobins. Bacteria were grown at 37°C and expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an absorbance of 0.6 at 600 nm. LB broth was to produce unlabeled proteins and M9 media for labelled proteins. M9 media contains 15NH4Cl and 13C6 glucose (Eurisotop) to label 15N and 15N/13C the proteins for NMR (Pham et al., 2018). Cells were harvested by centrifugation after 4 h of induction at 37° C and frozen at -80° C.

Solution nuclear magnetic resonance spectroscopy

We used NMR to complete the assignment of RodC as a first step to obtain its solution structure and to evaluate the purity, integrity and folding state of all purified proteins used in this study.

Assignment principle

To interpret NMR data (chemical shifts, relaxation rates, couplings, nuclear Overhauser effects…), it is necessary to first assign the signals (their resonance frequency expressed in ppm relative to a reference compound) to the atoms of the molecule. For proteins, the assignment strategy consists on first assigning the backbone and CB atoms using through-bond correlations based on J couplings of the 1H, 15N and 13C atoms of 15N/13C doubly labeled proteins. The most important atoms in this strategy are backbone 1H and 15N amide atoms. The spectrum that correlates both atom resonances is the 1H-15N HSQC (heteronuclear single quantum correlation experiment), or other variants (HMQC, SOFAST…). This spectrum, which contains one 1H and one 15N dimension, is a fingerprint of the structure and the dynamics of the protein. In an HSQC, each NH group normally generates one peak. Also, the tryptophan indole, asparagine and glutamine side-chain amides, and at ~low pH and/or ~low temperature arginine guanidinium and lysine amine resonances can be observed (Fig. 23).

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Assignment of NMR backbone chemical shifts of RodC

We prepared a RodC sample at a final concentration of 0.6 mM in 50 mM CD3COONa pH 4.5 and10% D2O. RodC experiments were run at 45 °C and recorded on 600-MHz or 800-MHz (Avance Neo, Bruker) proton resonance frequency spectrometers equipped with cryogenically cooled probes. Spectra were recorded using TopSpin 3.5.7 or 4.0 (Bruker), processed with NMRPipe (Delaglio et al., 1995) and analyzed with CCPNMR analysis 2.4.2 (Vranken et al., 2005).
Backbone and CB atoms of RodC resonances were assigned from 15N-1H HSQC, HNCO, HN(CO)CA, HNCACB, CBCA (CO)NH and HNHA experiments following standard procedures as published in Pham et al., 2018.

Secondary structure calculation

The secondary structure was predicted using the TALOS-N algorithm (Berjanskii and Wishart, 2005) from experimental chemical shifts (H, N, CO, CA, CB and HA) as described in (Pham et al., 2018). The TALOS-N (Torsion Angle Likeliness Obtained from Shifts) algorithm is based on the secondary structure information contained in backbone atom chemical shifts to predict torsion angles. The algorithm extensively uses neural networks to search for the best matches of chemical shifts and residue types (tripeptides) within a database of high-resolution XR structures and corresponding chemical shifts. To avoid predicting torsion angles for very flexible regions of proteins, TALOS-N uses the random coil index method developed by Berjanskii and Wishart (Berjanskii and Wishart, 2005), which predicts dynamics on the picosecond nanosecond time scale from chemical shifts.

Table of contents :

Preface
Acknowledgments
Résumé
Abstract
Table of contents
Index of figures
Index of tables
Abbreviations
Introduction 

1.1 Overview
1.2 The Fungal Kingdom
1.3 Fungal diseases in humans
1.4 Aspergillosis
1.5 Aspergillus fumigatus
1.5.1 Biology
1.5.2 Fungal Cell Wall
1.5.3 Aspergillus fumigatus Cell Wall
1.6 Pathobiology
1.7 Amyloid fibers
1.7.1 Functional and non-functional amyloids
1.7.2 How does the amyloid state form?
1.8 Hydrophobins
1.8.1 General aspects
1.8.2 Biological functions
1.8.3 Applications
1.8.4 Aspergillus fumigatus hydrophobins
1.9 Project description
Materials & Methods 
2.1 Proteins and peptides studied
2.2 Plasmid design
2.3 Expression and purification
2.4 Solution nuclear magnetic resonance spectroscopy
2.4.1 Assignment principle
2.4.2 Assignment of NMR backbone chemical shifts of RodC
2.4.3 Secondary structure calculation
2.5 Amyloid formation followed by ThT fluorescence
2.5.1 Effect of RodC point mutations on the fibrillation process
2.5.2 Effects of cell wall components on amyloid formation
2.6 Synchrotron radiation circular dichroism spectroscopy of hydrophobins
2.7 Fourier-transform infrared spectroscopy
2.8 Transmission electron microscopy
2.9 Atomic force microscopy
Results & Discussion
3.1 Chapter 1: Manuscript. Probing structural changes during self-assembly of surface-active hydrophobin proteins that form functional amyloids in fungi
3.1.1 Introduction
3.1.2 Framework and objective Manuscript
3.2 Chapter 2: Hydrophobin self-assembly
3.2.1 Introduction
3.2.2 A. fumigatus Class I hydrophobins form bona fide amyloids
3.2.3 Solution and ssNMR
3.2.4 Self-assembly kinetics of Rod hydrophobins
3.2.5 Effects of major fungal cell wall components on RodC self-assembly & perspectives
Bibliography

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