Protein- and Metal-dependent Interactions of a Prominent Protein in Mussel Adhesive Plaques

Abstract

The adhesive plaques of Mytilus byssus are investigated increasingly to determine the molecular requirements for wet adhesion. Mfp-2 is the most abundant protein in the plaques, but little is known about its function. Analysis of Mfp-2 films using the surface forces apparatus detected no interaction between films or between a film and bare mica; however, addition of Ca 2؉ and Fe 3؉ induced significant reversible bridging (work of adhesion W ad ≈ 0.3 mJ/m 2 to 2.2 mJ/m 2 ) between two films at 0.35 M salinity. The strongest observed Fe 3؉ -mediated bridging approaches the adhesion of oriented avidin-biotin complexes. Raman microscopy of plaque sections supports the co-localization of Mfp-2 and iron, which interact by forming bis-or tris-DOPA-iron complexes. Mfp-2 adhered strongly to Mfp-5, a DOPA-rich interfacial adhesive protein, but not to another interfacial protein, Mfp-3, which may in fact displace Mfp-2 from mica. In the presence of metal ions or Mfp-5, Mfp-2 adhesion was fully reversible. These results suggest that plaque cohesiveness depends on Mfp-2 complexation of metal ions, particularly Fe 3؉ and also by Mfp-2 interaction with Mfp-5 at the plaque-substratum interface.The mussel holdfast or byssus is emerging as an effective model system for studying the requirements for opportunistic underwater adhesion (1). The 3,4-dihydroxyphenylalanine (DOPA) 4 -rich mussel foot proteins (MFPs) of the adhesive footprint, namely Mfp-3 and Mfp-5, have been subjected to particular scrutiny (2-3) given the recent demonstration that DOPA is capable of mediating reversible adhesion to titania surfaces at forces of nearly 1 nanonewton/DOPA (4). Musselinspired catecholic polymers are engineered increasingly for moisture-resistant adhesive and coating applications (5-8).At least five other proteins known mostly as MFPs are present in the byssal plaque and contribute presumably to its adhesive performance: Mfp-1, Mfp-2, Mfp-4, Mfp-6, the prepepsinized collagens (9), and thread matrix protein ( Fig. 1) (10). Of these, only two have reasonably well established functions: Mfp-1 complexed to Fe 3ϩ provides a protective outer coating for the plaque and thread (11)(12), and the prepepsinized collagens are fiber-forming collagens that mediate the fusion of thread and plaque (13). Other proteins have been partially characterized and sequenced, but their role in byssal structure is largely speculative. Mfp-2 is particularly intriguing because it is the most abundant protein of byssal plaques comprising Ͼ25% of the plaque by weight (14). Mfp-2 has a mass of 45 kDa and consists of 11 tandem repeats of an EGF motif, each of which resembles a knot-like structure stabilized by three disulfide bonds (Fig. 2) (15-16). DOPA content is comparatively low at 3-5 mol % with an average of two residues per EGF repeat and three to four residues at the N-and C-terminal ends of Mfp-2. Surface binding studies of Mfp-2 using attenuated total reflectance Fourier transform infrared spectrometry showed the protein to be adsorbed rapidly to a variety of s...

Three intrinsically unstructured mussel adhesive proteins, mfp‐1, mfp‐2, and mfp‐3: Analysis by circular dichroism

“…23,36 Indeed, CD of mfp-2 in the far UV was almost identical to that of oxidized recombinant human EGF, 22 29,37 and can be disrupted by temperature or reduction of disulfides. SFA studies have independently shown that mfp-2 is Ca 2þ binding 7 and a partially Ca 2þ -bound form of mfp-2 was apparently used in the CD studies. Bound calcium on mfp-2 was not easily removed by chelation with excess EDTA at pH 5.5 but released with reduction of disulfide bonding [ Fig.…”

“…Previous studies allow the estimation of the hydrodynamic radius of mfp-2 at 3-4 nm. 7 In contrast to mfp-1, mfp-2, although also a flexible molecular necklace, has larger (40-45-res long) structured EGF domains.…”

“…Both proteins elute at about 22% acetonitrile. 7 Following HPLC, the proteins were freeze-dried and stored at À80 C.…”

“…Recruitment of the atomic force microscope, nanoindentation, and the surface force apparatus (SFA) has contributed much to understanding the physical and mechanical properties of mussel byssus and byssal proteins particularly the mfps. [5][6][7][8][9] However, structural studies at different length scales have been hampered by considerable challenges, including a menagerie of peculiar post-translational modifications, mfp sequence polymorphism, 2,10 the inability to crystallize any of the mfps, difficulty of purifying mg quantities, and the instability of mfps in solution. 11 This has lead to a variety of experimental approximations, which may or may not be biologically relevant.…”

High‐Performance Nanocomposites Inspired by Nature

“…[13,48] In addition, the 3,4-dihydroxyphenylalanine (DOPA)-rich proteins in the mussel byssal plaque, such as mfp-3 (20 mol% DOPA) and mfp-5 (28 mol% DOPA), coordinated with Fe 3+ , enable the plaque to adhere to rocks containing metal ions under water. [13,[49][50][51][52] In conclusion, mussel byssal displays multi functional features, such as self-healing and adhesion, with different types of metal coordination interactions.…”