A New Type Of Fibrous Protein And Its Regulative Function On Biomineralization

Bivalve ligament is a kind of natural organic-inorganic biocomposite materials that comprise of proteins and aragonite fibers. It functions to connect the two valves and serves to open them in living organism. When the abductor muscles of the organism contract and the valves close, elastic energy is stored in the ligament; when the abductor muscles relax, this energy causes the valves open. Therefore, bivalve ligament is also a kind of natural elastic biomaterials. Thoroughly studying on ligament structure, composition, and properties can provide not only scientific data, but also templates for synthesizing of high performance biomimetic materials. This article focus on ligaments of bivalve Solen grandis and Siliqua radiata, as compared with that of bivalve Sanguinolaria acuta, those of which are the common species from Beibu Gulf in South China. Firstly, microstructures and chemical compositions of these ligaments were investigated by scanning electron microscope, powder X-ray diffraction, Fourier transform infrared spectroscopy, and amino acid composition analysis; the forces which act on ligaments were also analyzed using the statics basic axioms. Then, main attentions and detailed studies were paid to the novel fibrous proteins discovered from S. grandis and S. radiata ligaments. The proteins were identified by matrix assisted laser desoiption ionization tandem time of flight mass spectrometry and liquid chromatography electrospray ionization tandem mass spectrometry, and their assembly mechanism were also analyzed. Using Fourier transform infrared spectroscopy, we determined the secondary structures of the proteins and analyzed their structure-function relationships. To investigate the mineralization mechanism of aragonite fibers in ligaments, biomimetic mineralization research was carried out using fibrous protein from S. radiata ligament as a template in vitro. Finally, the natural fluorescence of ligaments was preliminarily studied using fluorescence microscope and fluorescence spectrophotometer.Main conclusions of this work are drawn as follows:(1) We established a novel three layer structural model of bivalve ligament and found that aragonite fibers (with diameter of100-200nm) in inner layer of ligaments are formed by lots of attached anagonite nanoparticles. These fibers show two different orientations, i.e., fibers at dorsal part arrange from two laterals to central axis and orient posteriorly inclining almost parallel to dorsal surface; then, they turn gradually to ventral part and orient vertical to ventral surface. In addition, we first found that the accepted "growth lines" in inner layer of ligaments are formed by fractured "zigzag" aragonite fibers. These "zigzag" structures, we believe, result from the long-term repeated compressive stress acting on ligaments.(2) We discovered that fibrous protein Solenin and K58of S. grandis and S. radiata ligament, respectively, are a new type of fibrous proteins, and both of them are homologous to keratin type Ⅱ cytoskeletal1(KRT1) and type Ⅰ cytoskeletal9(KRT9). The proteins assemble initially by type Ⅰ and type Ⅱ monomers homologous to KRT9and KRT1, respectively, to form heterodimers in parallel manner; then, heterodimers join side by side in a staggered antiparallel manner to form tetramers; after that, heteropolymer intermediate filaments are formed gradually, and finally they self-assemble into protein fibers. We consider that these fibers are evolved to adapt the rapid burrowing life habits of those species.(3) Solenin and K58show remarkable expansibility and elasticity after absorbing water. These properties are coming mainly from the hydrogen bonds that formed between water molecules and hydrophilic amino acids in the protein, and from the "Glycine-loops" formed by "SGGG","SYGSGG","GGGGG", and "GGG" repeat sequences. While, the high β-sheet contents of Solenin (41.76%) and K58(46.20%) give the proteins excellent tensile strength and corrosion resistance properties.(4) With no additives and using K58as substrate, we obtained almost pure (95-100%) aragonite superstructures with lots of nanorods via transformation of ACC at ambient conditions. These nanorods, which are formed by attached aragonite crystals in the same crystallographic orientation, show morphology strikingly similar to aragonite fibers in ligaments. We believe the carboxyl groups of acidic amino acid residues of K58, which have short-range order similar to aragonite lattice, induce short-range order amorphous calcium carbonate (ACC) formation, resulting in the preferential transformation of ACC into aragonite. Meanwhile, we first propose that the formation of aragonite fibers in S. radiata ligament is controlled by K58.(5) We propose a new formation mode of aragonite nanorods and superstructures. Firstly, ACCs transform into disordered aragonite nanocrystals. Then, these crystals grow gradually, fuse with each other, rotate to get the same crystallographic orientation with driving force coming from the decrease of internal energy and removing of surface energy, and attach to form a nanorod. After that, colloidal ACCs attach to the rod in a2D maner by Brownian motion and evolve into more nanorods. Finally, aragonite superstructures with various morphologies are formed by continual aggregating of nanorods. This unconventional crystallization mode provides an important supplement for theory of crystallography.(6) We first discover that the inner layer of S. grandis, S. radiata, and S. acuta ligaments contain substances that can emit blue, yellow, green, and red fluorescence. The maximum excitation and emission wavelengths of these substances are380and467nm, respectively. We consider that this fluorescence is closely related to acid insoluble proteins in the inner layers of the ligaments, and it can be highlighted by Ca2+...