A novel Mfp-4 derived fusion tag enhances recombinant mussel foot protein production and facilitates purification.

Development of a cDNA microarray of zebra mussel ( Dreissena polymorpha) foot and its use in understanding the early stage of underwater adhesion

Event Abstract Back to Event A genetic and modular design strategy towards multi-functional and self-assembling underwater adhesives Chao Zhong1*, Mengkui Cui1 and Bolin An1 1 ShanghaiTech University, School of Physical Science and Technology, China Underwater adhesives with tunable compositions and functions have many important applications in both biomedical and marine fields, but remain a big challenge in current adhesive techniques. In previous study, we demonstrated a genetic modular strategy for engineering underwater adhesives based on the fusion of mussel foot proteins (Mfps) from Mytilus galloprovincialis with CsgA, the major subunit of adhesive curli fibers from Escherichia coli [1].​ However, these fibrous materials cannot satisfy all the requirements of ideal medical adhesives and their associated mechanism for underwater adhesion is also not fully understood. We utilize surface force apparatus (SFA) and atomic force microscopy (AFM) colloidal probe techniques, coupled with the genetic modular design strategy, to investigate the underwater adhesion mechanism of a variety of fusion proteins with tailor-designed protein domains. Furthermore, using ideal medical adhesives as blueprint, we leverage the genetic modular design strategy to further engineer biocompatible, injectable and ultra-strong adhesives based upon rational recombination of an amyloid-like gelation domain, cell adhesion moieties and mussel foot adhesive proteins. Collectively, this study will provide new insights into the adhesion mechanism of a new type of bio-inspired amyloid underwater adhesives and lay the foundation for engineering multi-compositional and multifunctional underwater adhesives.

Wound Healing In article number 2201108, Zheng Wang, Lin Wang, and co-workers develop a self-hydrophobized adhesive with robust underwater adhesion, resembling a mechanism underpinning the underwater adhesion of mussel foot proteins. This adhesive possesses multiple advantages outperforming commercial adhesives, such as in vivo wound healing-promoting effects, effective fluid leakage sealing, and rapid hemostasis activity.

Underwater adhesion is greatly desired in tissue transplantation, medical treatment, ocean transportation, and so on. However, common commercial polymeric adhesives are rather weakened and easily destroyed in water environment. In nature, some marine organisms, such as mussels, barnacles, or tube worms, exhibiting excellent underwater adhesion up to robust bonding on the rock of sea floor, can give exciting solutions to address the problem. Among these marine organisms, mussels exhibit unique underwater adhesion via the foot proteins of byssus. It has been verified that the catechol groups from the side chain of the mussel foot proteins is the main contribution to the unique underwater adhesion. Hence, inspired by the mussels’ underwater adhesion, many mussel‐mimetic polymers with catechol as end chains or side chains have been developed in the past decades. Here, we review recent progress of mussel‐inspired underwater adhesives polymers from their catechol‐functional design to their potential applications in intermediates, anti‐biofouling, self‐healing of hydrogels, biological adhesives, and drug delivery. The review may provide basis and help for the development of the commercial underwater adhesives.

Mussel foot protein with genetically incorporated hydroxyproline and DOPA exhibits enhanced phase separation and underwater adhesion.

Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly, and structure-function relationship of those natural amyloid fibers remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibers. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibers have an underwater adhesion energy approaching 20.9 mJ/m2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibers taken on their own at all pHs and exhibit better tolerance to auto-oxidation than Mfps at pH ≥7.0. This work establishes a platform for engineering multi-component self-assembling materials inspired by nature.

The exceptional underwater adhesion and self-healing capabilities of mussels have fascinated researchers for over two decades. Extensive studies have shown that these remarkable properties arise from a series of reversible and dynamic molecular interactions involving mussel foot proteins. Inspired by these molecular interaction strategies, numerous functional materials exhibiting strong underwater adhesion and self-healing performance have been successfully developed. This review systematically explores the nanomechanical mechanisms of mussel-inspired molecular interactions, mainly revealedby direct force measurement techniques such as surface forces apparatus and atomic force microscopy. The development of functional materials, including coacervates, coatings, and hydrogels, with underwater adhesion and self-healing properties, is then summarized. Furthermore, the macroscopic materialperformances are correlated with the underlying molecular mechanisms, providing valuable insights for the rational design of next-generation mussel-inspired functional materials with enhanced underwater adhesion and self-healing properties.

Since its invasion to the North American waters 20 years ago, the zebra mussel (Dreissena polymorpha) has negatively impacted the ecosystems through its firm underwater adhesion. The molecular mechanisms governing the functions of the zebra mussel byssus, the main structure responsible for maintaining the underwater adhesion, have received little attention. Our previously developed zebra mussel foot byssus cDNA microarray was applied in this study to identify the genes involved in different stages of the byssal threads generation. Byssal threads of zebra mussels were manually severed under laboratory conditions and the formation of new byssal threads was followed over a 3 week course. By comparing the gene expression profiles in different stages of byssal threads generation (byssogenesis) to their baseline values, we found that the number of unique byssus genes differentially expressed at 12-h, 1, 2, 3, 4, 7, and 21 days post-treatment was 13, 13, 20, 17, 16, 20, and 29, respectively. Comparisons were also made between two subsequent samples (e.g., 12 h vs. 1, 1 vs. 2 days, 2 vs. 3 days, and so on). Seven differentially expressed genes were selected for validation by using quantitative reverse transcription PCR (qRT-PCR) and the results were consistent with those from the microarray analysis. By using fluorescent in situ hybridization, we found that two microarray identified genes, BG15_F03-DPFP and BG16_H05-EGP, were expressed in two major byssus glands located in the zebra mussel foot: the stem-forming gland and plaque-forming gland, respectively. Moreover, the qRT-PCR of seven microarray identified genes with different zebra mussel samples suggested that they were also expressed in other mussel tissues beside the foot, albeit at much lower levels. This suggested that the microarray identified genes were produced primarily by the foot, and were likely associated with byssogenesis. The differentially expressed genes identified in this study indicated that multiple molecules are involved in byssogenesis, most likely performing multiple functions during the generation of byssal threads. These results obtained herein represent the first logical step toward understanding underwater attachment mechanisms employed by this invasive species.

Cation-π interaction is one of the most important noncovalent interactions identified in biosystems, which has been proven to play an essential role in the strong adhesion of marine mussels. In addition to the well-known catecholic amino acid, l-3,4-dihydroxyphenylalanine, mussel foot proteins are rich in various aromatic moieties (e.g., tyrosine, phenylalanine, and tryptophan) and cationic residues (e.g., lysine, arginine, and histidine), which favor a series of short-range cation-π interactions with adjustable strengths, serving as a prototype for the development of high-performance underwater adhesives. This work highlights our recent advances in understanding and utilizing cation-π interactions in underwater adhesives, focusing on three aspects: (1) the investigation of the cation-π interaction mechanisms in mussel foot proteins via force-measuring techniques; (2) the modulation of cation-π interactions in mussel mimetic polymers with the variation of cations, anions, and aromatic groups; (3) the design of wet adhesives based on these revealed principles, leading to functional materials in the form of films, coacervates, and hydrogels with biomedical and engineering applications. This review provides valuable insights into the development and optimization of smart materials based on cation-π interactions.

Mussels strongly adhere to a variety of surfaces by secreting byssal threads that contain mussel foot proteins (Mfps). Recombinant production of Mfps presents an attractive route for preparing advanced adhesive materials. Using synthetic biology strategies, we synthesized Mfp5 together with Mfp5 oligomers containing two or three consecutive, covalently-linked Mfp5 sequences named Mfp5(2) and Mfp5(3). The force and work of adhesion of these proteins were measured underwater with a colloidal probe mounted on an atomic force microscope and the adsorption was measured with a quartz crystal microbalance. We found positive correlations between Mfp5 molecular weight and underwater adhesive properties, including force of adhesion, work of adhesion, protein layer thickness, and recovery distance. DOPA-modified Mfp5(3) displayed a high force of adhesion (201 ± 36 nN μm-1) and a high work of adhesion (68 ± 21 fJ μm-1) for a cure time of 200 s, which are higher than those of previously reported Mfp-mimetic adhesives. Results presented in this study highlight the power of synthetic biology in producing biocompatible and highly adhesive Mfp-based materials.

The underwater adhesion of marine mussels is a fascinating example of how proteinaceous adhesives, although water-soluble to begin with, can be used in seawater. Marine mussels adhere to the substrate via adhesive plaques, where the adhesive proteins are located especially at the substratum's interface. One major compound of the adhesives in Mytilidae is the mussel foot protein 3b (mfp-3b). Here, recombinant mfp-3b (rmfp-3b) was produced in Escherichia coli. rmfp-3b showed upper critical solution temperature (UCST) mediated complex coacervation at pH 3.0 in the presence of citrate yielding a liquid-liquid phase separation. Further, the rmfp-3b coacervation could also be induced in seawater conditions such as the respective pH and ionic strength, but without UCST behavior. In particular, sulfate and citrate anions could significantly induce complex coacervation. This study provides insights into the molecular behavior of one of the key proteins of mussels involved in underwater adhesion and may inspire new applications of bioadhesives using recombinant mussel foot proteins.

Experimental test of the value of marine glue for ship building purposes

We report here that a dense liquid formed by spontaneous condensation, also known as simple coacervation, of a single mussel foot protein-3S-mimicking peptide exhibits properties critical for underwater adhesion. A structurally homogeneous coacervate is deposited on underwater surfaces as micrometer-thick layers, and, after compression, displays orders of magnitude higher underwater adhesion at 2 N m-1 than that reported from thin films of the most adhesive mussel-foot-derived peptides or their synthetic mimics. The increase in adhesion efficiency does not require nor rely on post-deposition curing or chemical processing, but rather represents an intrinsic physical property of the single-component coacervate. Its wet adhesive and rheological properties correlate with significant dehydration, tight peptide packing and restriction in peptide mobility. We suggest that such dense coacervate liquids represent an essential adaptation for the initial priming stages of mussel adhesive deposition, and provide a hitherto untapped design principle for synthetic underwater adhesives.

Facile biomimetic self-coacervation of tannic acid and polycation: Tough and wide pH range of underwater adhesives

Supramolecular assembly is commonly driven by noncovalent interactions (e.g., hydrogen bonding, electrostatic, hydrophobic, and aromatic interactions) and plays a predominant role in multidisciplinary research areas ranging from materials design to molecular biology. Understanding these noncovalent interactions at the molecular level is important for studying and designing supramolecular assemblies in chemical and biological systems. Cation-π interactions, initially found through their influence on protein structure, are generally formed between electron-rich π systems and cations (mainly alkali, alkaline-earth metals, and ammonium). Cation-π interactions play an essential role in many biological systems and processes, such as potassium channels, nicotinic acetylcholine receptors, biomolecular recognition and assembly, and the stabilization and function of biomacromolecular structures. Early fundamental studies on cation-π interactions primarily focused on computational calculations, protein crystal structures, and gas- and solid-phase experiments. With the more recent development of spectroscopic and nanomechanical techniques, cation-π interactions can be characterized directly in aqueous media, offering opportunities for the rational manipulation and incorporation of cation-π interactions into the design of supramolecular assemblies. In 2012, we reported the essential role of cation-π interactions in the strong underwater adhesion of Asian green mussel foot proteins deficient in l-3,4-dihydroxyphenylalanine (DOPA) via direct molecular force measurements. In another study in 2013, we reported the experimental quantification and nanomechanics of cation-π interactions of various cations and π electron systems in aqueous solutions using a surface forces apparatus (SFA).Over the past decade, much progress has been achieved in probing cation-π interactions in aqueous solutions, their impact on the underwater adhesion and cohesion of different soft materials, and the fabrication of functional materials driven by cation-π interactions, including surface coatings, complex coacervates, and hydrogels. These studies have demonstrated cation-π interactions as an important driving force for engineering functional materials. Nevertheless, compared to other noncovalent interactions, cation-π interactions are relatively less investigated and underappreciated in governing the structure and function of supramolecular assemblies. Therefore, it is imperative to provide a detailed overview of recent advances in understanding of cation-π interactions for supramolecular assembly, and how these interactions can be used to direct supramolecular assembly for various applications (e.g., underwater adhesion). In this Account, we present very recent advances in probing and applying cation-π interactions for mussel-inspired supramolecular assemblies as well as their structural and functional characteristics. Particular attention is paid to experimental characterization techniques for quantifying cation-π interactions in aqueous solutions. Moreover, the parameters responsible for modulating the strengths of cation-π interactions are discussed. This Account provides useful insights into the design and engineering of smart materials based on cation-π interactions.