Single-Molecule Force Spectroscopy Study on Modular Resilin Fusion Protein

Abstract

The adhesive and mechanical properties of a modular fusion protein consisting of two different types of binding units linked together via a flexible resilin-like-polypeptide domain are quantified. The adhesive domains have been constructed from fungal cellulose-binding modules (CBMs) and an amphiphilic hydrophobin HFBI. This study is carried out by single-molecule force spectroscopy, which enables stretching of single molecules. The fusion proteins are designed to self-assemble on the cellulose surface, leading into the submonolayer of proteins having the HFBI pointing away from the surface. A hydrophobic atomic force microscopy (AFM) tip can be employed for contacting and lifting the single fusion protein from the HFBI-functionalized terminus by the hydrophobic interaction between the tip surface and the hydrophobic patch of the HFBI. The work of rupture, contour length at rupture and the adhesion forces of the amphiphilic end domains are evaluated under aqueous environment at different pHs.

Figure 1

Schematic images and sizes of the studied modular proteins. (A) HFBI–dCBM consisting of the terminal hydrophobin HFBI (gray globular domain) and the CBMs (green globular domain) combined with polypeptide linkers. (B) dCBM–RLP–HFBI protein where the binding modules are combined with the resilin-like peptide (red random coil domain). (C) Cartoon of the force spectroscopy setup made up of an OTS-functionalized tip approaching on a cellulose-coated surface with the proteins casted on it. The sizes are estimated from the amino acid sequences (see Figure S1 and Tables S1 and S2).

Figure 2

(A) Schematic representation of the SMFS experiment: as the hydrophobic tip is approaching the surface, the hydrophobins will attach to the tip. (B) Tip retraction leads to elongation of the attached protein and eventual detachment of the protein from the tip. Typical force/distance curves, with fit included, measured for (C) HFBI–dCBM molecule, (D) dCBM–RLP–HFBI molecule at pH 5, and (E) at pH 11 and for the (F) cellulose surface at pH 5 and (G) at pH 11. On the right column, a schematic of the molecules/surfaces involved in the stretching is reported.

Figure 3

Histograms summarizing the (A) adhesion force, (B) contour length, (C) persistence length, and (D) work of rupture for HFBI–dCBM at pH 5 (black bars) and dCBM–RLP–HFBI at pH 5 (pink bars) and at pH 11 (red bars). The graphs (B,D) show the insets respectively for the contour length of HFBI–dCBM and the work of rupture of HFBI–dCBM and dCBM–RLP–dCBM at pH 5 to point out the distribution of the values in the lower range of x-axis.

Figure 4

QCM-D experiment on protein adsorption on the cellulose surface and consequent conformational changes. (A) Frequency change at pHs 5 and 11 on a cellulose-coated gold sensor (orange) and after injection (black arrow) of 0.1 mg mL⁻¹ HFBI−dCBM (red) and dCBM−RLP−HFBI (black) on the cellulose surface. (B) Dissipation and (C) thickness change during the same experiment.

Figure 5

AFM images of the (A) cellulose surface at pH 5; (B) HFBI–dCBM and (C) dCBM–RLP–HFBI drop-casted on the cellulose at pH 5; (D) cellulose surface at pH 11; and (E) HFBI–dCBM and (F) dCBM–RLP–HFBI drop-casted on the cellulose at pH 11. Insets represent a section analysis of the position indicated by the red line. The images were recorded on wet conditions under the respective buffer. Height range z from black to white is given in each image.

Figure 6

Schematic of different conformations of the resilin-like peptide at the detachment of the molecule. (A) At pH 5, resilin assumes a random coil conformation. (B) At pH 11, resilin assumes an extended conformation. (C,D) Attachment of multiple entangled resilin molecules at pH 11.

Figure 7

(A) Examples of approach curves on the cellulose surface at pH 5 (light blue dots) and pH 11 (dark blue small dots). (B) Examples of approach curves on the HFBI–dCBM layer at pH 5 (blue dots), dCBM–RLP–HFBI layer at pH 5 (violet small dots), and at pH 11 (light blue line).