Mussel adhesive protein provides cohesive matrix for collagen type-1α

Abstract

Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant load-bearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m(2)) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen.

Keywords: Collagen type-1; Mfp-3; Mussel foot proteins; Mylitus californiaus.

Figure 1

Studying interactions between collagen and the adhesive proteins from mussels. (a) In mussel byssal threads, collagens known as preCOLs mediate the transfer of load between the mussel plaque and the thread. PreCOLs come within a few nm of the mica surface, thus may bind directly to adhesive mfps such as mfp-3 (blue circles) and mfp-5 (green circles) [11]. (b) The symmetric SFA configuration used in this study to investigate collagen/mfp-3 interactions.

Figure 2

Quartz crystal microbalance in open cell mode showing relationship between adsorbed hydrated mass (calculated from Δfrequency) of mfp-3f, mfp-3s and COL1A1 to a TiO₂ surface and injection number. Injection number refers to the incremental addition of mfp-3 (C_mfp-3 = 20μg/ml) or COL1A1 (C_COL1A1 = 25μg/ml) aliquots, which are indicated by arrows for each protein. Note that COL1A1 did not adsorb to TiO₂. Adsorption solution conditions were 0.1 M sodium acetate buffer, pH 3.0 and 0.25 M KNO₃ at room temperature and pressure. For dissipation changes, see supporting data.

Figure 3

Mechanism of adsorption of COL1A1/mfp-3 to TiO₂. Mfp-3 is pre-adsorbed to TiO₂ before introducing COL1A1 (left), which adsorbs to surface-bound mfp-3. In contrast, COL1A1 by itself is not adsorbed to TiO₂ (right) but can be rescued for adsorption by mfp3-injection. Mfp-3 either binds to COL1A1 creating a sticky complex or to the TiO₂ surface.

Figure 4

The topography of mfp-3 on mica and COL1A1 and mfp-3 on mica was measured by AFM. (a) AFM topography image of mfp-3f on mica at pH 3.0. (b) and (c) show topography images of collagen type-1(COL1A1) deposited onto mica surface coated with mfp-3f in 0.1 M sodium acetate buffer, pH 3.0 and 0.25 M KNO₃. Expected dimensions of Type 1α tropocollagen i.e. ~1.5 nm x ~300 nm in agreement with the observed features (arrows).

Figure 5

Cohesion between two symmetric mfp-3f films with and without collagen. Representative force vs. distance plots for mcfp-3f deposited at C_mfp-3f = 20 μg/mL with collagen (C_COL1A1 = 25 μg/mL) injected between the two films in 0.1 M sodium acetate buffer, pH 3.0 and 0.25 M KNO₃.

Figure 6

Bridging cohesion in mfp-3 and the effect of pH and addition of COL1A1. (a) Representative force vs. distance plots for mfp-3f alone deposited at C_mfp-3f = 20 μg/mL in 0.1 M sodium acetate buffer, pH 3.0 and 0.25 M KNO₃. (b) Representative force vs. distance plots for mcfp-3f in 0.1 M phosphate buffer, pH 7.5 and 0.25 M KNO₃ from pH 3.0 and with collagen (C_COL1A1 = 25 μg/mL) injected between the two films in 0.1 M sodium acetate buffer, pH 3.0 and 0.25 M KNO₃.

Figure 7

H-bonding and cation-pi interactions between collagen and mfp-3 appear to mediate the strong but reversible binding between these molecules. Collagen type-1 deposited onto mfp-3 pre-adsorbed mica surface in 0.1 M sodium acetate buffer, pH 3.0 and 0.25 M KNO₃.