# Effect of Nanohydroxyapatite on Silk Fibroin-Chitosan Interactions--Molecular Dynamics Study
## 3 Results and Discussion
Of course, the impact of deacetylation on the affinity of chitosan for fibroin requires further investigation. Another interesting aspect to explore is the ratio of fibroin to chitosan. In this study, a relatively large number of fibroin chains were used compared to chitosan, allowing for extensive involvement of chitosan in molecular contacts, as seen in Figure 2. Notably, in the case of composites containing fibroin, chitosan, and nanohydroxapatite, increasing the proportion of fibroin is advantageous for the development of osteogenic cells [87]. The analysis of molecular contacts involving Ca2+ seems to offer valuable insights into the stabilization of potential biomaterials used for bone regeneration. It is well known that the addition of calcium salts is advantageous for the solubilization of fibroin [17], indicating Ca2+/peptide interactions. When considering the impact of Ca2+ ions on the peptide structure, it is important to note that they can strongly interact with fibroin, leading to a reduction in the number of \(\beta\)-sheet domains [112]. This, in turn, affects the mechanical properties of the material. Notably, an experimental study by [PERSON] et al. [113] confirms that the carboxylic groups in ASP and GLU in silk peptides form ionic contacts with Ca2+ and K+, which has a significant impact on their rheological properties. However, the presence of chitosan has a beneficial effect on \(\beta\)-sheet formation [67; 111; 114],  
Figure 5: Visualization of silk fibroin helices interacting with chitosan in the presence of acetic acid. Hydrogen bonds are denoted by pink dotted lines, while ionic contacts are denoted by solid red lines. Solid green lines stand for hydrophobic contacts and solid blue pi-stacking. Fibroin helices are presented as solid turquoise lines, whereas chitosan is represented by a ball-stick model with colors representing the following elements: carbon—turquoise, nitrogen—dark blue, phosphorus—yellow, oxygen—red, and hydrogen—white. (**a**) shows interactions involving tyrosine (TYR) and aspartic acid (ASP) residues, while (**b**) illustrates interactions involving alanine (ALA) and serine (SER) residues. which probably compensates the effect of Ca\({}^{2+}\) ions. As expected, in light of the this research, the interactions with Ca\({}^{2+}\) can be observed in the regions characterized by \(\beta\)-sheet conformation (Figure 6c,d). Structural details of the identified ionic bridges (ASP-Ca\({}^{2+}\)-ASP, ASP-Ca\({}^{2+}\)-GLU, ASP-Ca\({}^{2+}\)-GLY, GLU-Ca\({}^{2+}\)-GLU, GLU-Ca\({}^{2+}\)-GLY) are provided in Supplementary Table S3. To identify hydrogen bonds, a default YASARA protocol based on an energy calculation with a specified threshold of \(-6.25\) kJ/mol was applied [115]. The molecular dynamics simulations revealed that ALA, ARG, ASN, ASP, GLN, GLU, GLY, LEU, PRO, SER, THR, TYR, and VAL form the strongest hydrogen bonds, whether the complexes are stabilized by CH\({}_{3}\)COOH or Ca\({}^{2+}\)/HPO\({}_{4}\)\({}^{2-}\). The highest binding energy values were observed in the case of ARG, reaching 22.30 \(\pm\) 3.45 kJ/mol for acetic-acid-stabilized complexes and 19.49 \(\pm\) 5.86 kJ/mol for systems stabilized by Ca\({}^{2+}\) and HPO\({}_{4}\)\({}^{2-}\) ions. Among these hydrogen bonds, interactions formed by ASP were found to be the most significant contributors, constituting approximately 26.3% of all hydrogen bonding interactions. A considerable contribution to complex stabilization was also noted for SER residues. In the case of complexes stabilized by acetic acid, hydrogen bonds involving SER exhibited a binding energy of 18.16 \(\pm\) 5.45 kJ/mol, accounting for 10.5% of all hydrogen bonds, while those stabilized by Ca\({}^{2+}\) and HPO\({}_{4}\)\({}^{2-}\) ions accounted for 10% (18.36 \(\pm\) 5.56 kJ/mol). Additionally, THR residues were observed to form relatively strong hydrogen bonds, with binding energies of 17.72 \(\pm\) 5.91 kJ/mol (CH\({}_{3}\)COOH) and 18.80 \(\pm\) 5.60 kJ/mol (Ca\({}^{2+}\)/HPO\({}_{4}\)\({}^{2-}\)).