Gelatin, a multifunctional protein produced by means of collagen hydrolysis, exhibits key physicochemical properties, such as gelation, emulsifying properties and biodegradability, and is widely used in food, healthcare, pharmaceuticals, cosmetics, and other fields [1,2]. The basic physicochemical properties of gelatin depend on many factors, including the source of material and protein amino acid sequence. Fish gelatin (FG) has emerged as a promising alternative to mammalian gelatin, offering advantages such of biocompatibility, a lower risk of zoonotic disease transmission, and compliance with specific religious dietary laws. Furthermore, its production contributes to the valorization of waste from the fishing industry, which can account for up to 30 % of processing byproducts [3,4]. However, a significant drawback of FG is its inferior gelling strength and lower gelation and melting temperatures compared to its mammalian counterparts [5,6].

One approach to enhance the natural gelling properties of fish gelatin is the structural modification of gelatin hydrogels by the introduction of polysaccharides of various natural origins. Many polysaccharides themselves have gelling properties, which are widely used in bioprinting, drug delivery systems, and in other fields [7]. For example, chitosan is used in wound dressings and nerve regeneration due to its inherent antibacterial properties, biocompatibility, and ability to be processed into porous scaffolds [8]. Alginate is a cornerstone material in 3D bioprinting and cell encapsulation due to its gentle gelation with divalent cations, creating hydrogels that mimic the extracellular matrix [9]. Iota-carrageenan is used in drug delivery and recently explored in tissue regeneration for its ability to form strong, thermo-reversible gels [10].

Extensive experimental studies have explored the use of polysaccharides like carrageenans, sodium alginate, and chitosan to improve FG's functional and rheological properties [[11], [12], [13]]. Combination of polysaccharides with gelatin increases their scaffold properties, which are particularly relevant in regenerative medicine, bioprinting and other fields [11,12]. The functional efficacy of these materials in applications such as cell adhesion, growth factor retention, and tissue regeneration depends partially on the interactions between gelatin and the polysaccharide [12,13]. By varying the polysaccharides and modification methods, it is possible to obtain hydrogels with specified properties [6,13].

While interactions of FG with polysaccharides have been extensively studied, most of existing researches are focused on bulk hydrogel properties [[14], [15], [16], [17], [18], [19], [20], [21], [22]], the atomistic details of these complexes remain poorly understood. This knowledge gap arises from fundamental difficulties in computational representation of gelatin properties. Gelatins are characterized by a low net charge and uneven distribution of charged and uncharged amino acid residues along polypeptide chain. The large molecular weight (>100 kDa), non-globular, flexible structure of gelatins and heterogeneous charge distribution complicate the utilization of all-atomic simulations for modeling of protein structure and gelatin-ligand interactions. Previous studies used the fragments of gelatin molecules or collagen-like peptides to analyze the gelatin/collagen-ligand interactions [[23], [24], [25]]. However, we assume that the use of short collagen-like peptides cannot reflect well the gelatin binding behavior, while heterogeneous charge distribution can play a key role in stability and dynamics of gelatin molecules and in their interaction with ligands. The irregular charge distribution along gelatin chains creates the local regions with varying charge density, forming small binding sites that can attract both anionic and cationic polysaccharides.

In this work, we employ two in silico approaches - molecular docking and molecular dynamics (MD) simulations to investigate FG interaction with anionic (iota-carrageenan and alginate) and cationic (chitosan) polysaccharides at atomic resolution. To elucidate the role of charge distribution we designed four FG model fragments with distinct net charges and charge distribution. Our study evaluates binding affinities, complex stability, and the influence of FG charge heterogeneity on polysaccharide binding. We demonstrate that charge distribution is critical for polysaccharide binding, and anionic and cationic polysaccharides form stable complexes with FG. Unlike prior empirical studies, our computational approach provides insights into dynamics and structure of complexes with estimation of relative contributions of electrostatic, hydrophobic, and hydrogen-bonding forces. This work advances the rational design of the FG-based biomaterials by establishing structure-function relationships at molecular level.