Single-Molecule Force Spectroscopy Study on Modular Resilin Fusion Protein

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.

. Modular protein consisting of random coils (blue) connected with extended linkers (red). The diameter of the random coil modules where calculated from the radius of gyration RG taking N as the number of amino acids and 0.36 nm as the length of one amino acid l.

Force-distance curves
More examples of force-distance curves (Fd) of the set up with the OTS-tip and the proteincellulose-coated SiO2 surface are reported in Figure S2 in order to show additional data and conformations, forces, and energies that the molecules can reach. For example, the resilin at pH 5 can stretch till 100 nm having different conformations (B). At pH 11, the contour length have its maxima values around 140 nm and some curves are observed even at 150-200 nm (C). Fd curves are also presented for cellulose at pH 5 and 11(D). Figure S2. Examples of force distance curves recorded having a silanized tip with octadecyltrichlorosilane (OTS) and a protein coated surface. The graphs A) and B) report the force / distance curves for HFBI-dCBM and dCBM-RLP-HFBI at pH 5, respectively, whereas graphs C) reports the ones for dCBM-RLP-HFBI at pH 11. D) Control experiment with OTS coated tip done on cellulose at pH 5 and 11 without proteins.

Force distance curves: reversed set up
In Figure S3, the reversed set up (protein-cellulose coated-tip and OTS functionalized SiO2 surface) is represented. Examples of the force-distance profiles measured during detachment of the HFBI-dCBM and dCBM-RLP-HFBI with a cellulose-coated tip mounted on a cantilever having a spring constant of 0.03 N/m are shown in Figure S3 B and C. Typically, in the retraction curve (red), a large adhesion peak in the nN range is obtained and located at 10-20 nm from the surface, followed by two or three smaller peaks further away from the surface. The separation of the peaks and their magnitude depend on the studied protein, both being larger for the dCBM-RLP-HFBI than for the HFBI-dCBM. The peak of the main interest was the last one that represents the final linkage between the tip and the OTS-Si surface created by single or a few molecules only. As the tip retracts from the surface, there is a progressive detachment of the molecules adsorbed on the cellulose layer: When the protein-coated tip is withdrawn, not all the molecules detached simultaneously but the number of them decreased as the cantilever-substrate distance increases ( Figure S3 B and C).
The total work of rupture is represented by the area under the retraction curves and consists of two main factors 1) the deformation of the layer when compressed between the tip and the surface and 2) adhesion of the protein layer to the hydrophobic surface. When assuming an elastic deformation of the layer, the adhesion energy is given as the hysteresis between the approach and the retraction part. This value is large for the protein coated cellulose tip and the hydrophobized surface, indicating that the protein coated cellulose has strong adhesion to the hydrophobized surface, indicating that the hydrophobins are pointing out from the cellulose coating as sketched in Figure  S3 A. The blue curve refers to the progressive approach of the tip that snaps in contact with the surface as the small step is observed in the curve. The analysis of the data was carried out separately for the large peak representing adhesion of multiple molecules and for the last peak representing single molecules. Figure S3. A) Schematic representation of the attachment/detachment process of proteins from the surface of interest during the approach (green arrow) and withdraw (brown arrow) steps. Increasing the tip-surface separation during the tip retraction the number of molecules interacting is reduced. Force / distance curves recorded using the protein coated tip on the OTS-surfaces for B) HFBI-dCBM and C) dCBM-RLP-HFBI. D) Control experiment of the cellulose coated tip on OTS-SiO2. Arrows mark the evaluated peaks: the large adhesion peak representing adhesion of multiple molecules and the last peak representing single molecules. Figure S4 shows the adhesion force of the large adhesion peak (A) and the work of rupture (B) of both protein constructs at pH 5. The adhesion force is taken as the maximum value of the force in the retraction curve in the experiments. The work of rupture was obtained by integrating the area under the retraction curve and represents the work needed for detaching the tip from the substrate. The data show that the distribution of adhesion force and work of rupture for the large adhesion peak are quite similar for both the fusion proteins. This is an interesting finding in view of applications, such as using monolayers of these proteins as adhesives.

Properties of the molecular protein layers
The rupture force of the single molecules analysed from the last peak of the force curve yielded similar values for the rupture force as the experiments where the protein was immobilised on the surface instead of the tip. This provided more certainlty about the assumed orientation and detachment mechanism of the molecules. The further analysis of the peaks was, however, challenging due to the presence of the overlapping force peaks and fitting to the WLC model resulted in unrealistic contour lengths. Therefore, the data obtained by this set up was not included in the full data sets. Figure S4. Results of the SMFS experiment with protein functionalised tip. The data represents the large peak on the force curves. Histograms of A) the adhesion force as the maximum force of the large adhesion peak and B) work of rupture.

QCM-D experiments
The Voigt model was applied to analyze the measured changes in frequency Δf, and dissipation ΔD and in order to extract information on layer thickness whereas the mass of the bound proteins was estimated by the application of the Sauerbrey equation Where C (17.7 ng cm -2 Hz -1 at f0 = 5 MHz) is the mass sensitivity constant and n is the overtone number. The Sauerbrey equation is valid for rigid, evenly distributed and sufficiently thin adsorbed films. Here, the Sauerbrey mass was used as an estimate of the bound amount of the proteins.

AFM Images of the proteins
The morphology of the protein coated surfaces was investigated by AFM as reported in Figure  S5. Figure S5. AFM images of hydrophobin fusion proteins on cellulose coated SiO2 surfaces. The pictures represent a comparison of the two fusion proteins A, C) HFBI-dCBM and B, D) dCBM-RLP-HFBI. The cellulose structure dominates the appearance. All the images are recorded in wet conditions under sodium acetate buffer. Total z-scale from black to white is given in each image.
Plain cellulose at pH 11 Figure S6 reports the histograms characterizing the energies, forces, and length of the cellulose at pH 11. Peaks with few hundreds of E/KT and pN, and 10-30 nm of length are observed. Such parameters are attributed to the stretching of some cellulose filaments that are easily pulled at pH 11 compared to the cellulose at pH 5. Figure S6. Histograms summarizing the A) rupture force, B) work of rupture, C) contour length and D) persistence length for the plain cellulose at pH 11.

Entanglement of resilin fusion proteins
At pH 11, dCBM-RLP-HFBI proteins showed a much broader adhesion force distribution than at pH 5 assumed to be caused by a more extended conformation and a resulting entanglement of the proteins. This assumption was drawn from a closer analysis of the data obtained from the comparison with the worm like chain model, which is presented in the following. In Figure S7A, the adhesion force FA is plotted against the contour length LC. Every point represents a single experiment. For pH 5 (red circles), the adhesion force is constant for all evaluated contour lengths with a large standard derivation as described earlier. For pH 11, however, three different populations can be distinguished: One, for which FA is nearly constant for different LC with very low scattering (blue circles), and two other ones, where FA depends nearly linearly on LC (blue up and down triangles with purple and green edges). In the Figure  It is therefore reasonable to interpret the data from population 3 as resulting from entangled protein chains. In this interpretation the degree of entanglement will then determine the observed contour and persistence length. Moreover, the number of proteins involved in the entanglement and the number of possible attachment points, which are likely not only the hydrophobin end-domains, will determine the observed adhesion force. In this model, population 1 with its very uniform adhesion force will then correspond to stretching events of individual chains. Population 2 in Figure S7 A, show a similar behavior as population 3, only shifted to higher contour length values, and in Figure S7 B persistence length values in between the ones from population 1 and 3.
These data might therefore be interpreted as resulting from events where proteins are attached to the tip and not to the surface but instead entangled with other surface attached proteins. This situation might result e.g. from previous events where chains are picked up by the AFM tip, are still attached to this and are then entangled with individual chains on the surface. The fact, that data in population 2 are less numerous than in the other populations supports this assumption.

Figure S7
A) Adhesion force vs. contour length from the evaluation of the force spectroscopy data. Each data point represents a single stretching event of dCBM-RLP-dCBM proteins at pH 5 (red circles) and pH 11 (blue value). For pH 11, different populations can be identified. B) Rupture force vs. persistence length for the same data as in A. The different populations are also marked here.
Therefore, we assume for experiments at pH 11 an entanglement of the proteins, which might lead to a high number of attachment points in close vicinity. Breaking one anchor will then directly cause the other(s) to break, which might be undistinguishable from a single breaking event.
Purification and expression of dCBM-RLP-HFBI Figure S9. Design of the pMIs124 expression construct. The expression cassette integrates into the cbh1 gene locus via the cbh1 promoter and cbh1 3′ flanking sequences. The CBHI carrier, Kex2 cleavage site, double cellulose binding modules (dCBM), resilin-like polypeptide (RLP), hydrophobin I (HFBI), and hygromycin resistance cassette (HygR) parts are represented in the figure.  Figure S10. A) Expression of dCBM-RLP-HFBI determined via immunoblot detection with antistrep tag antibody. The culture supernatant from day 5 was diluted in LSB loading buffer so that 0.7 µl could be loaded in a 20 µl volume along with the standard protein corresponding to 200, 100, 50, 25 ng. B) SDS PAGE gel of the purified dCBD-RLP-HFBI protein strained with Gel-Code blue Coomassie stain.

Characterization of TMSC and cellulose surfaces
In order to test the conversion from TMSC to cellulose, XPS, AFM, ellipsometry, and water contact angle measurements have been carried out as reported in Figure S11 and in Table S4.
A) B) S12 Figure S11. Top row: XPS data (C-1s) for a monomolecular film of thrimethylsilylcellulose spin coated at 5000 rpm on SiO2 (A) before and (B) after HCl treatment showing strongly reduced C-Si contributions. Bottom row: AFM pictures of SiO2 substrate spin coated with 1 mg/mL of cellulose before C) and after D) the exposition to HCl vapours. The morphology confirms the hydrolysis reaction. The spin coating procedure and the morphological characterization by AFM (not shown) have been previously performed at highest concentration in order to choose the most homogeneous and flat layer. Water contact angle and thickness for TMSC 10 mg/mL and 5 mg/mL spin coated on SiO2 before and after conversion into cellulose are shown in Table S4.