Coil dimensions of the mussel adhesive protein Mefp-1

doi:10.1016/j.biomaterials.2004.04.032

Biomaterials

Volume 26, Issue 11, April 2005, Pages 1231-1236

https://doi.org/10.1016/j.biomaterials.2004.04.032 Get rights and content

Abstract

To obtain a better understanding of factors controlling cross-linking rates of Mussel adhesive proteins, we study the conformation of the Mussel Adhesive Protein Mefp-1. The dimensions of Mefp-1 in solution are determined by dynamic light scattering. Under physiological conditions, the hydrodynamic radius R_H of Mefp-1 is found to be 10.5±1.1 nm. Measured Mefp-1 dimensions are compared with theoretical dimensions of Mefp-1 in random coil conformations. We have strong indications that Mefp-1, under dilute and physiological conditions, has a self-avoiding random walk conformation with helix-like deca-peptide segments. With a number of segments of approximately 90, the segment length is found to be 2.7 nm.

Introduction

Mussels like the common blue mussel Mytilus edulis are able to glue themselves underwater to a variety of (hydrophilic) surfaces by using a mix of proteins. Until now, six adhesive proteins, varying in type and molecular weight, have been isolated from the phenolic glands of the blue mussel's foot [1]. They are indicated by Mefp-1 to 6, the abbreviation of M. edulis foot protein. Common for all these proteins is the presence of Dopa (3,4-dihydroxyphenylalanine).

Already in an early stage, the potential of mussel adhesive proteins (MAPs) as high performance medical adhesive was recognised [2], [3], [4]. However, because the yield from extraction is low, several attempts have been made to produce large amounts of proteins using molecular biology based methods [5], [6] or to make synthetic mimics of MAPs, which resulted in various peptide-based products [7], [8], [9], [10]. More recently, the focus has changed to the use of various polymer backbones to which Dopa groups are attached [11], [12], [13], [14].

The main challenge in these bio-mimetic approaches is to sufficiently simplify the structure to enable easy-to-use synthesis routes, while keeping the essential elements of the MAPs. An additional problem is that by mimicking one molecule, it is not taken into account that the mussel uses a mix of proteins, with different molecular weights and amino acid composition.

In mussel adhesion, the interplay between the rate of deposition and the cross-linking rates determines the properties of the adhesive bond [15]. Cross-linking rates determine curing times. Especially in medical applications these must be well controlled.

The kinetics of reactions of macromolecules bearing reactive groups are unusual in many respects. They not only depend on the chemical reaction rates, but also on the dynamics and the conformation of the macromolecules. Important parameters are, for example, solvent quality, molecular weight and chain stiffness [16]. Therefore, in the design of synthetic mimics of MAP, information of the conformation of MAPs and the effect of molecular weight on cross-linking rates are indispensable.

In a previous paper, we determined the reaction rates of Dopa groups in the mussel adhesive protein Mefp-1 [17]. In this paper, we determine the chain stiffness under physiological conditions. The possible conformation of Mefp-1 is discussed by comparing the experimental results with literature data.

Section snippets

Mefp-1

Mefp-1 has a molecular weight of 115 kDa based on mass spectroscopy [18] and of 130 kDa on the basis of gel chromatography [19]. It is mainly composed of a repeating uniform deca-peptide. One Mefp-1 molecule contains 71 deca-peptides and 12 hexa-peptides of comparable composition, as shown in Fig. 1. The distribution of the deca- and hexa-peptides over the Mefp-1 backbone is illustrated in Fig. 2. The total number of amino acids is 897.

Looking at the molecular structure of Mefp-1, several

Coil dimensions

In the cross-linking kinetics for random coiled molecules, the reaction rate depends on the reactivity of the groups and the conformation of the coil. For example, in dilute systems in a good solvent the effective rate constant for a reactive group connected to a chain segment with length A is given by [16] $k≃k_{0} A^{3} N ̃^{−vg}$ in which k₀ is the reactivity of a single segment, Ñ an effective chain length, related to the location on, and number of segments (N) in the chain [16], [17]. The exponent $ν= 35$ .

Experimental

The samples of Mefp-1 had a purity of 95% and were delivered in 1% citric acid at concentrations of 1.3 mg/ml. These stock solutions were stored in the dark at 4°C. To prevent oxidation of DOPA groups, a 50 mmol l(+)-ascorbic acid (Fluka) buffer in millipore water was used. NaCl was added for the desired ionic strength and the pH was set at 3.8. The DLS apparatus was equipped with a JDS Uniphase 633 nm 35-mW laser, an ALV sp-125 s/w 93 goniometer, a fibre detector and a Perkin Elmer photon counter

Results

First, measurements were performed with Mefp-1 concentrations varying from 0.013 to 0.13 mg/ml. No considerable effect of the Mefp-1 concentration on the measured dimensions was detected. Therefore, in further analysis, there was no need for extrapolations to zero concentration, and all subsequent measurements were done with Mefp-1 concentrations of 0.13 mg/ml. In Fig. 3 the thus obtained hydrodynamic radii of Mefp-1 are plotted as a function of the inverse ionic strength, enabling an

Analysis and discussion

Sedimentation studies on Mefp-1, performed by Deacon et al. [22], resulted in a frictional ratio f/f₀ of 3.2±0.3 at pH=4.5 and ionic strength of 0.1 m, with f₀ as the friction coefficient of the dehydrated protein, and a partial specific volume of dehydrated Mefp-1 of 0.75 ml/g. This frictional ratio corresponds with an R_H (of the hydrated coil) of 10±1.0 nm and agrees well with our DLS results.

Conclusions

The experimentally obtained hydrodynamic radius of Mefp-1 of 10.5±1 nm can be explained by two different models. Assuming that Mefp-1 is a fully random protein, it can be described as a random polymer in θ-conditions, which is also called a Gaussian chain. If it is assumed that within the deca-peptide segment there is some helical-like structure present, the found hydrodynamic radius can only be explained by assuming good solvent conditions. The reactivity of the Dopa groups and the hydrophilic

Acknowledgements

The authors thank M. Qvist (Biopolymer Products AB, Alingsås, Sweden) who kindly supplied the Mefp-1 samples and the Dutch Technology Foundation STW for their financial support.

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Therefore, on the basis of the unique adhesive and anti-corrosion properties it presenting and the environmental consideration, Mefp-1 will be a promising choice for an adhesive film preparation on Mg-1.0Ca alloy. Mefp-1 is primarily composed of repeating deca-peptide and hexa-peptides, and contains a high percentage of hydroxylated amino acids: DOPA (dihydroxyphenyl alanine), diHYP (dihydroxy proline) and HYP (hydroxy proline) [27]. DOPA is recognized as a crucial functional role in the adhesive and cohesive properties of Mefp-1 [31,32].
A range of ex situ and in situ analytical techniques were applied to gain insights into the formation and properties of the pre-formed Mefp-1 film on magnesium-1.0 wt.% calcium (Mg-1.0Ca) alloy. The results revealed that the Mefp-1 film pre-formed at pH 4.6 shows a net-like structure, whereas it is more packed at pH 8.5. in situ scanning micro-reference electrode technique results demonstrated the Mefp-1 films formed at both pH can effectively inhibit the localised corrosion of Mg-1.0Ca alloy. Moreover, the film pre-formed at pH 4.6 provides an increasing corrosion inhibition to Mg-1.0Ca alloy during 7 days of exposure.
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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 Fe³⁺ 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 Fe³⁺ 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 Fe³⁺, mfp-1 (Mc) contains Dopa with two distinct Fe³⁺-binding tendencies and prefers to form intramolecular complexes with Fe³⁺. In contrast, mfp-1 (Me) is better adapted to intermolecular Fe³⁺ binding by Dopa. Addition of Fe³⁺ 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.
Salt- and pH-induced desorption: Comparison between non-aggregated and aggregated mussel adhesive protein, Mefp-1, and a synthetic cationic polyelectrolyte
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It was found that most aggregates have a RH value of around 20 nm, which should be compared with the value of about 9 nm found for the non-aggregated sample. The latter value is consistent with literature data [23]. The aggregation of Mefp-1 is suggested to be promoted by high concentration in the stock solution.
Mussel adhesive proteins are of great interest in many applications due to their ability to bind strongly to many types of surfaces under water. Effective use such proteins, for instance the Mytilus edulis foot protein – Mefp-1, for surface modification requires achievement of a large adsorbed amount and formation of a layer that is resistant towards desorption under changing conditions. In this work we compare the adsorbed amount and layer properties obtained by using a sample containing small Mefp-1 aggregates with that obtained by using a non-aggregated sample. We find that the use of the sample containing small aggregates leads to higher adsorbed amount, larger layer thickness and similar water content compared to what can be achieved with a non-aggregated sample. The layer formed by the aggregated Mefp-1 was, after removal of the protein from bulk solution, exposed to aqueous solutions with high ionic strength (up to 1 M NaCl) and to solutions with low pH in order to reduce the electrostatic surface affinity. It was found that the preadsorbed Mefp-1 layer under all conditions explored was significantly more resistant towards desorption than a layer built by a synthetic cationic polyelectrolyte with similar charge density. These results suggest that the non-electrostatic surface affinity for Mefp-1 is larger than for the cationic polyelectrolyte.
Adsorption of Mefp-1: Influence of pH on adsorption kinetics and adsorbed amount
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Mussel adhesive proteins have received considerable attention due to their ability to bind strongly to many surfaces under water. Key structural features of these proteins include a large number of 3,4-dihydroxyphenyl-l-alanin (DOPA) and positively charged lysine residues. We elucidate the effects of solution pH, in the pH range 3–9, on adsorption kinetics, adsorbed amount, and layer structure on silicon oxynitride by employing Dual Polarization Interferometry. As a comparison, the cationic globular protein lysozyme was also investigated. The zeta-potential of the silicon oxynitride substrate was determined as a function of pH, and the isoelectric point was found to be below pH 3. Mefp-1 is positively charged at pH < 10, and thus, the protein is expected to have an electrostatic attraction for the surface at all pH values investigated. The adsorbed amount and the initial adsorption rate were found to increase with solution pH, and no significant desorption occurred due to rinsing with pure water. The layer thickness after rinsing was 3–4 nm, except at pH 3, where the adsorption was limited to a small amount. Covalent cross-linking of the Mefp-1 layer with NaIO₄ resulted in a small but significant compaction and increase in refractive index of the layer. The results are discussed in terms of the role of DOPA and electrostatic interactions for the adsorption of Mefp-1 to silicon oxynitride.
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Mfp-1 is moderately large (~ 110 kDa in M. edulis) and highly repetitive with over 70 tandem repeats of the decapeptide sequence [A-K-P-Y˚-S-O⁎-O-T-Y⁎-K] in which O, O⁎, Y˚ and Y⁎ denote 4-hydroxyproline, 3,4-dihydroxyproline, usually tyrosine but occasionally Dopa, and always Dopa, respectively (Filpula et al., 1990; Taylor et al., 1994a,b) (Fig. 7A). Despite the highly conserved repeat motif, the protein is an extended random coil in solution (Deacon et al., 1998; Haemers et al., 2005). Addition of Fe3+ to mfp-1 in solution at pH 7 results in the formation of highly stable coloured complexes: red at high Dopa to Fe3+ ratios and purple at ratios of roughly 1:1 (Taylor et al., 1996) (Fig. 7B).
The conventional wisdom regarding sclerotization in arthropod exoskeletons is that tissue stiffness and polyphenolic content are tightly coupled. Recent progress on the biochemistry and mechanics of sclerotized structures from non-arthropod invertebrates such as mussels, polychaetes and squid suggests that this premise is too simple and needs to be more closely scrutinized. Emerging mechanistic insights about structure–property relationships in sclerotized extracellular composites include the following: (1) tissue stiffness can be significantly adjusted by the relative organization and density of rigid versus flexible domains in the protein components (mussel byssal thread); (2) structures can be rich in polyphenols without being sclerotized (mussel adhesive plaque); (3) stiffness and hardness can be adjusted by the relative abundance of interactions between protein-based imidazole and catecholate ligands and transition metals (polychaete jaws and mussel cuticle); (4) polyphenol-derived covalent cross-link density in a protein–chitin composite offers some stiffening, but gains from incrementally controlled dehydration are far greater (squid beak); and finally, (5) protein domains, ligand and metal densities, covalent cross-linking and dehydration are elaborately manipulated to create stiffness gradients between mechanically mismatched tissues.

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Coil dimensions of the mussel adhesive protein Mefp-1

Abstract

Introduction

Section snippets

Mefp-1

Coil dimensions

Experimental

Results

Analysis and discussion

Conclusions

Acknowledgements

Trends Biotechnol

Biomaterials

Tetrahedron

Arch Biochem Biophys

Biomaterials

Prog Biophys Mol Biol

Adv Colloid Interf Sci

Comput Phys Commun

Biophys J

J Biol Chem

Adv Prot Chem

Methods Enzymol

Adhesion à la Moule

Integrative Comparative Biol

Les colles chirurgicales en ophtalmologie

J Fr Ophtalmol

Development of a microbial system for production of mussel adhesive protein

Expression of a model peptide of a marine mussel adhesive protein in Escherichia coli and characterization of its structural and functional properties

J Polym Sci Pol Chem

Synthesis and wettability characteristics of model adhesive protein sequences inspired by a marine mussel

Biomacromolecules

Synthetic polypeptide mimics of marine adhesives

Macromolecules

Synthesis of the repeating deca-peptide unit of Mefp1 in orthogonally protected form

J Org Chem

Chemical modification of soy protein for wood adhesives

Macromol Rapid Commun