# Effect of Nanohydroxyapatite on Silk Fibroin-Chitosan Interactions--Molecular Dynamics Study
## 1 Introduction  
It is widely recognized that the interactions between proteins and polysaccharides are pivotal in both living organisms [1] and the development of novel biomaterials [2]. Among the polysaccharide analogues, proteoglycans deserve special attention. These biomolecules are composed of protein and glycosaminoglycan (GAG) moieties bound to the protein by the serine and threonine fragments [3]. They are typically found on the cell surface and in the extracellular matrix (ECM). Proteoglycans are essential for the strength and flexibility of cartilage and tendons. The absence of proteoglycans is associated with heart and respiratory failure, defects in skeletal development, and increased tumor metastasis [4]. Silk threads are considered to be one of the strongest natural fibers. They exhibit a high tensile strength of 4.8 GPa, very good flexibility [5, 6, 7] and high thermal stability [8, 9, 10]. Silk is more resistant to loads than Kevlar (the most potent manufactured fiber to date) [7, 11, 12]. Spiderweb silk exhibits exceptional mechanical properties [10; 13]. However, silk produced on an industrial scale is primarily sourced from domesticated mulberry silkworms (_B. mori_) due to their ease of cultivation and higher thread yield from their cocoons compared to spider webs [10; 13]. Natural silk thread is composed of two tightly packed bundles of fibroin fibers, glued with a sericin sheath and soluble in hot water. Fibroin protein is a dominant component of raw silk, accounting for 70%-80% of its weight [14; 15; 16]. The other components are sericin, wax, fats, and dyes [12; 17]. Fibroin, has a high content of amino acids, with serine comprising approximately 12% [18], whereas collagen contains four times less amino acids [19]. The repetitive sequence of amino acids (alanine and glycine) already in the primary structure makes silk fibroin a highly ordered protein. An essential repeating motif of the fibroin amino acid sequence is -(GLY-SER-GLY-ALA-GLY-ALA)\({}_{\text{n}}\). These chains, connected by hydrogen bonds, form the antiparallel \(\beta\)-sheet structure. Due to the periodic primary structure, there are glycine residues in the secondary structure on one side of the surface of the \(\beta\)-sheet, interacting with the glycine residues of the adjacent chain. Similarly, on the other side of the surface of the \(\beta\)-sheet, there are serine and alanine residues interacting with similar residues of another neighboring chain (Figure 1). This quasi-crystalline structure is interrupted by stretches of the sequence containing other amino acids (e.g., TYR, ASP, ARG and VAL). Silk fibers with a high proportion of regular glycine-alanine fragments tend to be more flexible due to weak interactions between chains in the \(\beta\)-sheet structure, but less stretchable as the polypeptide chains are under significant tension in this secondary structure. Fiber properties such as gloss and softness depend on differences in the size and characteristics of amino acid residues within irregular structural fragments. Crystalline regions within  
Figure 1: Silk thread morphology, based on [10; 20; 21; 22]. The letters A, B and C denote crystalline region, \(\alpha\)-helix structure and non-oriented amorphous area, respectively. fibers, marked as A in Figure 1, consist of tightly packed motifs of the antiparallel \(\beta\)-sheet, aligned along the fiber axis. Dispersion angle measurements revealed the presence of crystallites with dimensions of 2 \(\times\) 2 \(\times\) 2 nm [20]. However, these dimensions increase to 5-7 nm in length at slower reeling speeds [23]. Additionally, the analysis identified non-crystalline regions with two distinct morphologies: (i) oriented regions with an \(\alpha\)-helix structure (B, Figure 1) and (ii) non-oriented amorphous areas (C, Figure 1).