Nadine R. Martinez Rodriguez

A mussel-derived one component adhesive coacervate

Marine organisms process and deliver many of their underwater coatings and adhesives as complex fluids. In marine mussels one such fluid, secreted during the formation of adhesive plaques, consists of a concentrated colloidal suspension of a mussel foot protein (mfp) known as Mfp-3S. The results of this study suggest that Mfp-3S becomes a complex fluid by a liquid-liquid phase separation from equilibrium solution at a pH and ionic strength reminiscent of the conditions created by the mussel foot during plaque formation. The pH dependence of phase separation and its sensitivity indicate that inter-/intra-molecular electrostatic interactions are partially responsible for driving the phase separation. Hydrophobic interactions between the non- polar Mfp-3S proteins provide another important driving force for coacervation. As complex coacervation typically results from charge-charge interactions between polyanions and polycations, Mfp-3S is thus unique in being the only known protein that coacervates with itself. The Mfp-3S coacervate was shown to have an effective interfacial energy of ⩽1mJm(-2), which explains its tendency to spread over or engulf most surfaces. Of particular interest to biomedical applications is the extremely high adsorption capacity of coacervated Mfp-3S on hydroxyapatite.

Interfacial pH during mussel adhesive plaque formation

Mussel (Mytilus californianus) adhesion to marine surfaces involves an intricate and adaptive synergy of molecules and spatio-temporal processes. Although the molecules, such as mussel foot proteins (mfps), are well characterized, deposition details remain vague and speculative. Developing methods for the precise surveillance of conditions that apply during mfp deposition would aid both in understanding mussel adhesion and translating this adhesion into useful technologies. To probe the interfacial pH at which mussels buffer the local environment during mfp deposition, a lipid bilayer with tethered pH-sensitive fluorochromes was assembled on mica. The interfacial pH during foot contact with modified mica ranged from 2.2 to 3.3, which is well below the seawater pH of ~ 8. The acidic pH serves multiple functions: it limits mfp-Dopa oxidation, thereby enabling the catecholic functionalities to adsorb to surface oxides by H-bonding and metal ion coordination, and provides a solubility switch for mfps, most of which aggregate at pH ≥ 7–8.

Bridging Adhesion of Mussel-Inspired Peptides: Role of Charge, Chain Length, and Surface Type

The 3,4-dihydroxyphenylalanine (Dopa)-containing proteins of marine mussels provide attractive design paradigms for engineering synthetic polymers that can serve as high performance wet adhesives and coatings. Although the role of Dopa in promoting adhesion between mussels and various substrates has been carefully studied, the context by which Dopa mediates a bridging or nonbridging macromolecular adhesion to surfaces is not understood. The distinction is an important one both for a mechanistic appreciation of bioadhesion and for an intelligent translation of bioadhesive concepts to engineered systems. On the basis of mussel foot protein-5 (Mfp-5; length 75 res), we designed three short, simplified peptides (15–17 res) and one relatively long peptide (30 res) into which Dopa was enzymatically incorporated. Peptide adhesion was tested using a surface forces apparatus. Our results show that the short peptides are capable of weak bridging adhesion between two mica surfaces, but this adhesion contrasts with that of full length Mfp-5, in that (1) while still dependent on Dopa, electrostatic contributions are much more prominent, and (2) whereas Dopa surface density remains similar in both, peptide adhesion is an order of magnitude weaker (adhesion energy Ead ∼ −0.5 mJ/m2) than full length Mfp-5 adhesion. Between two mica surfaces, the magnitude of bridging adhesion was approximately doubled (Ead ∼ −1 mJ/m2) upon doubling the peptide length. Notably, the short peptides mediate much stronger adhesion (Ead ∼ −3.0 mJ/m2) between mica and gold surfaces, indicating that a long chain length is less important when different interactions are involved on each of the two surfaces.

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

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.

Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein-6

Mytilus foot protein type 6 (mfp‐6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp‐6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp‐6 showed high purification yields of 5–6 mg L−1 cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp‐6.1 protein showed high 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano‐Newton range, we show that rmfp‐6.1 is also able to rescue the oxidation‐dependent adhesion loss of mussel foot protein 3 (mfp‐3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4‐dihydroxyphenyl‐l‐alanine in mfp‐3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp‐6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage.

Tough Coating Proteins: Subtle Sequence Variation Modulates Cohesion

Mussel foot protein-1 (mfp-1) is an essential constituent of the protective cuticle covering all exposed portions of the byssus (plaque and the thread) that marine mussels use to attach to intertidal rocks. The reversible complexation of Fe(3+) by the 3,4-dihydroxyphenylalanine (Dopa) side chains in mfp-1 in Mytilus californianus cuticle is responsible for its high extensibility (120%) as well as its stiffness (2 GPa) due to the formation of sacrificial bonds that help to dissipate energy and avoid accumulation of stresses in the material. We have investigated the interactions between Fe(3+) and mfp-1 from two mussel species, M. californianus (Mc) and M. edulis (Me), using both surface sensitive and solution phase techniques. Our results show that although mfp-1 homologues from both species bind Fe(3+), mfp-1 (Mc) contains Dopa with two distinct Fe(3+)-binding tendencies and prefers to form intramolecular complexes with Fe(3+). In contrast, mfp-1 (Me) is better adapted to intermolecular Fe(3+) binding by Dopa. Addition of Fe(3+) did not significantly increase the cohesion energy between the mfp-1 (Mc) films at pH 5.5. However, iron appears to stabilize the cohesive bridging of mfp-1 (Mc) films at the physiologically relevant pH of 7.5, where most other mfps lose their ability to adhere reversibly. Understanding the molecular mechanisms underpinning the capacity of M. californianus cuticle to withstand twice the strain of M. edulis cuticle is important for engineering of tunable strain tolerant composite coatings for biomedical applications.

Peptide Length and Dopa Determine Iron‐Mediated Cohesion of Mussel Foot Proteins

Mussel adhesion to mineral surfaces is widely attributed to 3,4-dihydroxyphenylalanine (Dopa) functionalities in the mussel foot proteins (mfps). Several mfps, however, show a broad range (30-100%) of Tyrosine (Tyr) to Dopa conversion suggesting that Dopa is not the only desirable outcome for adhesion. Here, we used a partial recombinant construct of mussel foot protein-1 (rmfp-1) and short decapeptide dimers with and without Dopa and assessed both their cohesive and adhesive properties on mica using a surface forces apparatus (SFA). Our results demonstrate that at low pH, both the unmodified and Dopa-containing rmfp-1s show similar energies for adhesion to mica and self-self interaction. Cohesion between two Dopa-containing rmfp-1 surfaces can be doubled by Fe3+ chelation, but remains unchanged with unmodified rmfp-1. At the same low pH, the Dopa modified short decapeptide dimer did not show any change in cohesive interactions even with Fe3+. Our results suggest that the most probable intermolecular interactions are those arising from electrostatic (i.e., cation-π) and hydrophobic interactions. We also show that Dopa in a peptide sequence does not by itself mediate Fe3+ bridging interactions between peptide films: peptide length is a crucial enabling factor.

Correction to "Tough Coating Proteins: Subtle Sequence Variation Modulates Cohesion".

Addition/Correction pubs.acs.org/Biomac Correction to “Tough Coating Proteins: Subtle Sequence Variation Modulates Cohesion” Saurabh Das, Dusty R. Miller, Yair Kaufman, Nadine R. Martinez Rodriguez, Alessia Pallaoro, Matthew J. Harrington, Maryte Gylys, Jacob N. Israelachvili,* and J. Herbert Waite* Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 10, 2015 | http://pubs.acs.org Publication Date (Web): June 17, 2015 | doi: 10.1021/acs.biomac.5b00759 Biomacromolecules, 2015, 16 (3), 1002−1008. DOI:10.1021/bm501893y F igure 3 caption corrected. Corrections highlighted in bold. Figure 3. pH dependence of Fe 3+ -mediated cohesion between two symmetric mfp-1 (Mc) films. Representative force vs distance plot showing the interaction between two symmetric mfp-1 (Mc) films deposited at 50 μg/mL in 0.1 M sodium acetate buffer, pH 5.5, 0.25 M KNO 3 , and 1 mM bis− tris with C Fe 3+ = 0 (gray) and 10 μM (blue) at pH 5.5. The cohesion between the mfp-1 (Mc) films was preserved after increasing the pH to 7.5 (magenta). (b) Representative force vs distance plot showing the interaction between two symmetric mfp-1 (Me) films deposited at 20 μg/mL in 0.1 M sodium acetate buffer, pH 5.5, 0.25 M KNO 3 , and 1 mM bis−tris with C Fe 3+ = 0 (gray) and 10 μM (blue) at pH 5.5. The surfaces showed a weak bridging cohesion (W c < 0.2 mJ/m 2 ) after increasing the pH to 7.5 (magenta). It should be noted that C Fe 3+ is the concentration of ferric cation in the bulk solution between the surfaces. Flushing with buffer at pH 7.5 removes iron from the bulk solution, however, to the extent that the preadsorbed protein films already had some bound Fe 3+ , the Dopa−Fe 3+ complexes will be present in them. Published: June 17, 2015