Protein aggregation into amyloid fibrils is a significant phenomenon linked to several diseases, including Alzheimer's disease, Parkinson's disease, and type 2 diabetes, through both systemic mechanisms and localized amyloid deposition [[1], [2], [3], [4]]. These amyloid fibrils are important hallmarks of protein misfolding diseases in both the central nervous system and peripheral tissues. In particular, insulin amyloid formation contributes to insulin-derived amyloidosis (IDA), which occurs in diabetic patients receiving repeated insulin injections. This process is also suspected to play a role in insulin resistance associated with type 2 diabetes [5,6]. Moreover, in pharmaceutical production, insulin fibrillation poses a challenge for large-scale peptide preparation. Lowering the pH to the range of 2–4 during manufacturing promotes aggregation, which can lead to reduced biological activity and elicit undesired immune responses in patients [[7], [8], [9]]. These fibrils are stable, insoluble structures that are mainly formed by β-sheet stacking and hydrogen bond networks. The formation of fibrils is influenced by several environmental and structural factors, such as temperature, ionic strength, mechanical stress, and pH. Under suitable conditions, even proteins that are typically soluble can undergo conversion into amyloid structures [[10], [11], [12], [13], [14], [15]].

Insulin is a small peptide hormone that plays a crucial role in regulating blood glucose levels in the body. It is composed of two polypeptide chains, A and B, which are linked together by disulfide bonds. The native structure of insulin is mainly α-helical, and in its storage form, it exists as a hexamer stabilized by zinc ions. Insulin is synthesized and released by the β-cells of the pancreas, facilitating the utilization and storage of glucose, amino acids, and fatty acids. In the bloodstream, the hexamer naturally dissociates into dimers and then into active monomers, which are essential for its biological function [[16], [17], [18], [19]]. However, in acidic or destabilizing environments, this dissociation can occur prematurely or in an uncontrolled manner, resulting in monomers or dimers that are more susceptible to aggregation [20]. The fibrillation of insulin has been extensively investigated by several experimental and theoretical methods [[21], [22], [23]]. It has been shown that under acidic conditions, insulin readily forms amyloid fibrils with a high content of β-sheets. The conversion from α-helical to β-sheet-rich structures occurs during the nucleation phase, followed by elongation and saturation. The hydrophobic core of the insulin fibril is mainly composed of residues from the B-chain, including the LVEALYL segment, which forms steric zipper arrangements and stabilizes the structure. Studies have shown that mutations in this region can alter the aggregation behavior and stability of the fibril. Unlike Aβ fibrils, which exhibit well-characterized polymorphs such as LS-shaped structures, insulin fibrils are known to display structural polymorphism, and their morphology strongly depends on environmental conditions such as pH, ionic strength, temperature, and even the method of fibrillation induction [[24], [25], [26], [27], [28]]. Among the various environmental parameters, pH has emerged as a key factor affecting both the formation and long-term stability of insulin fibrils. At low pH values, the overall net charge of the protein is altered due to protonation of acidic side chains, leading to a reduction in electrostatic repulsion and promoting close packing of the chains within the fibril [29]. Conversely, at neutral or slightly basic pH, deprotonation of glutamate and aspartate residues can increase electrostatic repulsion, destabilizing the fibril [30,31]. Experimental studies, such as those by Darussalam et al. [32] and Yoshihara et al. [2], have shown that insulin fibrils initially formed at low pH undergo progressive structural disintegration when exposed to pH 7. These studies reported a decrease in β-sheet content and an increase in structural disorder at physiological pH, highlighting the destabilizing effect of deprotonation on fibril integrity. These findings emphasize that insulin fibrils are not consistently stable across varying pH conditions, a property that contrasts with other amyloids, such as Aβ fibrils [24,26].

Several experimental studies have examined the effect of pH on insulin fibril morphology [[33], [34], [35]]. Utilizing spectroscopic techniques such as Fourier-transform infrared (FTIR) spectroscopy, circular dichroism (CD), and Raman spectroscopy, researchers have shown that the secondary structure and compactness of fibrils vary with pH. One study [30] demonstrated that insulin fibrils formed at low pH gradually lost their ordered structure when exposed to neutral pH. In another study, Kurouski et al. [36] used Raman spectroscopy to show that specific vibrational signatures associated with β-sheets were more intense in fibrils formed at acidic pH, indicating a more stable and ordered arrangement. Although these experimental studies provide valuable macroscopic insights into insulin fibril behavior, their resolution is limited, often focusing on general aspects such as fibril formation or disassembly without addressing residue-specific hydrogen bonding, inter-monomer contacts, or the distribution of interactions over the fibril core. Furthermore, relatively few studies have applied MD simulations to systematically compare the behavior of preformed insulin fibrils under different pH conditions, despite the demonstrated utility of this approach in related systems [31,37,38]. Although in vitro fibrils may differ from their in vivo counterparts, they serve as essential models for exploring the biophysical principles of amyloid stability.

MD simulations provide atomic-level insight into fibril stability, enabling real-time observation of hydrogen bonds, salt bridges, backbone flexibility, and residue-specific interactions. Becker et al. [26] performed atomic-resolution simulations on Aβ fibrils, demonstrating how pH-dependent deprotonation of side chains can disrupt salt bridges and reorganize hydrogen bond networks. Similarly, Gspöner et al. [39] investigated the early aggregation steps of an amyloid-forming peptide from the yeast prion Sup35, revealing that side-chain hydrogen bonding and aromatic stacking play a critical role in stabilizing in-register parallel β-sheet arrangements. In the case of tau fibrils, a recent MD study combining replica exchange molecular dynamics (REMD) and steered MD has been instrumental in uncovering conformational flexibility and aggregation pathways in solution and membrane environments [40]. Collectively, these findings indicate that MD simulations enable direct observation of residue-level interactions over time and complement experimental approaches by providing atomistic insights that experiments alone often cannot resolve [41].

The current study focuses on utilizing MD simulations to investigate the structural and dynamic properties of insulin amyloid fibrils under varying pH conditions (acidic at pH 2 and neutral at pH 7). The fibril model consisted of ten stacked insulin monomers derived from a previously validated experimental structure [31]. For each pH condition, three independent simulations were performed, resulting in a total of six production runs (Fig. 1). The primary objective was to assess how fibril stability and dynamics differ between these two distinct pH environments and to identify specific residues or regions that are critical for maintaining structural integrity. This work provides a comparative, atomic-level analysis of the pH-dependent behavior of insulin fibrils, integrating data from multiple simulations and aligning these findings with existing experimental results. By elucidating the molecular mechanisms that govern fibril stability and disruption, the research offers valuable insights into insulin aggregation processes. The research results highlighted here could play an essential role in advancing our comprehension of physiological processes and enhancing pharmaceutical innovation, ultimately contributing to the development of more robust insulin formulations or effective inhibitor designs to mitigate unwanted fibril formation.