Molecular Dynamic Study on the Structure and Thermal Stability of Mutant Pediocin Pa-1 Designed to Contain Extra Cystein Residues

doi:10.21203/rs.3.rs-3416062/v1

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Molecular Dynamic Study on the Structure and Thermal Stability of Mutant Pediocin Pa-1 Designed to Contain Extra Cystein Residues

https://doi.org/10.21203/rs.3.rs-3416062/v1

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published 01 Mar, 2024

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Pediocin and pediocin-like bacteriocins are antimicrobial compounds used in the food industry owing to their remarkable thermal stability and resistance to heat. Enhancing their stability at elevated temperatures and harnessing a bacteriocin derivative molecule produced through such improvements holds promise not only in the food industry but also within the pharmaceutical sector due to their broad-spectrum potential. Although previous studies have examined the two disulfide bonds present in Pediocin PA-1, there has been no inclination to increase the number of disulfide bonds. This study aimed to design mutant peptides by introducing a third disulfide bond, in addition to the two naturally occurring ones in pediocin PA-1, and assess the thermal stability of these mutant peptides. In this study, five mutant peptides (Mut 1, Mut 2, Mut 3, Mut 4, Mut 5) with dual Cys mutations were designed. Initially, the thermal stability of these peptides across a range of temperatures (298–394 K) was evaluated through 100 nanoseconds of molecular dynamics simulations. Subsequently, the most stable mutant peptides (Mut 1, Mut 4, and Mut 5) were selected for further investigation. Molecular dynamics simulations lasting 300 nanoseconds were conducted and analyzed to observe the thermal stability of these structures. The analysis encompassed parameters such as RMSD, RMSF, DSSP, and SASA. The stabilization of these structures was compared with Pediocin PA-1, chosen as the reference molecule. In silico analysis revealed that the free Cys residues did not form a third disulfide bond. Furthermore, most mutants (Mut 1, Mut 5), including the reference structure, exhibited a loss of their helical structure, which is known to be crucial for antimicrobial activity, and the disruption of one of the naturally occurring disulfide bonds at high temperatures. Conversely, Mut 4 retained two disulfide bonds even at elevated temperatures, preserved its helical structure, and demonstrated a thermal stability profile similar to that of Pediocin PA-1. The findings of this study suggest that Mut 4, a highly stable peptide, may represent an exceptionally heat-resistant antimicrobial alternative, contingent upon further analysis to confirm its activity.

Pediocin PA-1

thermal stability

disulfide bonds

MD Simulations

GROMACS

Cys residues

Bacteriocins, ribosomally synthesized by various bacterial species, possess potent antimicrobial properties against pathogenic bacteria [1–5]. In recent years, bacteriocins have garnered increasing significance, primarily due to their versatile applicability in both the health and food industries. The demand for bacteriocins, particularly in the food sector, stems from the growing need for pathogen-free, naturally-sourced products with fewer chemical preservatives [1, 5–7]. These antimicrobial peptides have proven to be exceptional food preservatives within the food industry. Some key attributes that underscore their importance include their resistance to pH variations and heat, their ability to inhibit foodborne pathogens even at low concentrations, and their susceptibility to deactivation by intestinal proteases [7]. Additionally, in a world increasingly concerned about the emergence of antibiotic-resistant bacterial strains, the quest for antimicrobial alternatives to conventional antibiotics, particularly in the healthcare domain, amplifies the significance of bacteriocins [1, 2, 5, 8].

Bacteriocins are categorized into four distinct classes, with Class IV earning the designation of "bacteriolysis" due to the presence of complexes that contain carbohydrate and lipid fragments. Excluding Class IV, it can be established that bacteriocins are primarily classified into three major classes [9]. Class I encompasses bacteriocins that feature post-translational modifications, while Class II includes those without post-translational modifications or those adopting a cyclic structure. Meanwhile, Class III is comprised of heat-sensitive and large-sized bacteriocins [1, 9].

Pediocin and pediocin-like bacteriocins fall under the category of Class IIa bacteriocins. Typically, pediocins consist of 37–48 amino acids, exhibit anti-listerial properties, display heat resistance (up to 121°C), and possess peptide structures that incorporate one or two disulfide bonds [8, 10, 11]. These bacteriocins are primarily produced by lactic acid bacteria (LAB), especially by pediococci and lactobacilli species [5]. They share a highly conserved peptide region known as the "pediocin box," which is located in the highly conserved N-terminal region [1, 8, 10, 12–14]. The amino acid sequence in the C-terminal region of these bacteriocins may vary among different bacteriocin variants. Pediocin PA-1/AcH stands as the most well-known member of these bacteriocins, boasting a heat resistance of 80°C and beyond. It is produced by LAB and ranks as one of the most extensively studied bacteriocins, following nisin [4, 14, 15].

Pediocin PA-1/AcH boasts a structural arrangement featuring two vital disulfide bonds, specifically Cys9-Cys14 and Cys24-Cys44. It is well-documented that the disulfide bond, particularly in the C-terminal region, plays a pivotal role in the antimicrobial activity of this bacteriocin [8, 10, 14, 16]. Furthermore, it is a known fact that bacteriocins lacking disulfide bonds in this specific region exhibit antimicrobial activity that is significantly diminished, with a reduction of 30–50 times at 37°C when compared to pediocin PA-1 [10]. Moreover, the second disulfide bond within the C-terminal region serves as an effective safeguard, preserving the structural integrity of the peptide even when exposed to high temperatures [13]. This phenomenon is further supported by the correlation between temperature-related activity and the optimal growth temperatures of bacteria that produce pediocin.

In recent times, in silico methods, notably molecular dynamics studies, have become increasingly prevalent. They serve the purpose of elucidating the structural characteristics of peptides and gathering data that may be challenging to acquire through traditional wet lab experiments, particularly in the field of pharmaceuticals. Molecular dynamics (MD) studies represent a category of in silico techniques employed to simulate the atomic-level movements of target molecules, adhering to the principles of biophysics within specified conditions [17]. These MD studies have proven to be invaluable tools for investigating conformational changes in molecules and interactions with ligands. They enable simulations at the femtosecond timescale, rendering results obtained through this method highly desirable for interpreting various biological processes [17, 18].

The present study employed MD simulations to delve into the impact of amino acid sequence mutations in pediocin PA-1 on peptide stability across varying temperatures. Furthermore, building upon the knowledge that human defensins typically involve three to six disulfide bonds in mutation processes [19], we also explored the potential for introducing a third disulfide bond in bacteriocins like pediocin. To achieve this, we initiated modifications to the peptide by introducing cysteine (Cys) residues. These residues possess the capacity to enhance the stability of the secondary structure when they are in their free form and also facilitate the formation of disulfide bonds. In pursuit of the potential for disulfide bond formation and an overall enhancement in structural stability, we implemented dual mutations within the peptide, involving double Cys substitutions. Subsequently, we subjected these peptides to MD simulations over a temperature range spanning from 298 to 394 K (equivalent to 25 to 121°C). The objective was to investigate the thermal stability of the peptides and gauge the impact of additional Cys residues, potentially leading to the formation of disulfide bonds. This study puts forth a novel peptide, designed to mimic the stabilization observed in the structure of pediocin PA-1, with the aim of showcasing its antimicrobial activity in vitro. This holds particular relevance for high-temperature studies, paving the way for potential applications in this domain.

Obtaining the three-dimensional structure of Pediocin PA-1 and constructing mutant molecules

The three-dimensional structure of Pediocin PA-1 was obtained using the crystallographic data available in the Protein Data Bank (PDB) with the corresponding PDB code: 5UKZ, serving as the reference for pediocin's 3D structure. To construct mutant peptide structures, PyMOL version 2.5 [20] was employed. Subsequently, an analysis of the potential effects of these mutations on the structure and functionality of the peptides was conducted. This analysis was facilitated by the HOPE server [21].

MD Simulations

For the process of structure minimization, GROMACS 2022.3 [22–24] was employed, and this operation was executed on a MacBook Pro equipped with a 2.3 GHz 8-Core Intel Core i9 processor. Throughout the MD simulations, the OPLS-AA force field [25, 26] was utilized during the minimization phase. The water model of choice was TIP3P [27], and the system was configured within a rhombic dodecahedron box at a 1 Angstrom (Å) unit. MD simulations were carried out using the computational resources of TÜBİTAK ULAKBİM's High Performance and Grid Computing Center (TRUBA), where the GROMACS 2022.3 program [22–24] was already integrated into the system. To obtain initial data and refine the structures, MD simulations were conducted at various temperatures (298, 310, 313, 323, 333, 343, 348, 353, 363, 373, 394 K; equivalent to 25, 37, 40, 50, 60, 70, 75, 80, 90, 100, 121°C). These simulations employed the same force field-water model and were run for a duration of 100 nanoseconds (ns). Following this initial simulation, structures that exhibited stability, as confirmed by Root Mean Square Deviations (RMSD), Root Mean Square Fluctuations (RMSF), and data pertaining to the secondary structure of proteins (DSSP), were selected for the subsequent MD simulations. The primary simulation was designed to last for 300 ns. To analyze the simulation data, the resources available at TRUBA included VMD (Visual Molecular Dynamics) version 1.9.2 [28], while PyMOL version 2.5 [20] was utilized for visualizing the molecular structures. During the examination of the MD simulation results, graphical representations of RMSD, RMSF, DSSP, and Solvent Accessible Surface Area (SASA) were generated, and these results were subsequently interpreted.

Evaluation of the Physicochemical Structure in HOPE Server and SASA analysis after MD Simulation of the mutant peptides

The pediocin molecule initially exists in a precursor form known as "pre-pediocin," where it incorporates a signal peptide comprising 62 amino acids (as depicted in Fig. 1A). Notably, pre-pediocin remains inactive and lacks the critical disulfide bonds between Cys-Cys necessary for its bacteriocidal activity. Activation occurs through the cleavage of the 19-amino acid signal peptide in the pre-pediocin molecule, a process catalyzed by proteases within the cytoplasm. This cleavage results in the transformation of the remaining 44 amino acid peptide structure into an exponentially active state. Within the active peptide, specific amino acid segments play pivotal roles in structuring its functionality. Amino acids located at positions 4–6 (GNG) and 39–41 (QGN) within the active peptide adopt a "turn" conformation. Additionally, amino acids positioned at 7–9 (VTC) and 14–16 (CSV) contribute to the formation of a "-sheet" structure. Furthermore, the amino acids spanning the 18–35 region, recognized as the active site of pediocin, adopt a "-helix" structure, thus shaping the peptide's functional structure (as illustrated in Fig. 1A and 1B) [8].

In this research, the Pediocin PA-1 molecule served as the reference structure, with the PDB Code: 5UKZ, and was analyzed using PyMOL. This analysis was particularly focused on assessing its impact on the "-helix" structure within the N-terminal region and examining potential disulfide bonds. To introduce amino acid mutations and convert them into Cys residues, modifications were carried out. It is well-established that free Cys residues within the peptide structure, which have not engaged in disulfide bond formation, can influence the activity and thermal stability of the macromolecule. Simultaneously, these unbound Cys amino acids can contribute to stabilizing the "-helix" structure [29, 30]. To this end, five distinct mutations were introduced into the amino acid sequence of Pediocin PA-1, involving the replacement of various amino acid residues with Cys amino acids. A detailed account of these mutations is available in Table 1. Subsequently, Mutation 1 (Mut 1), Mutation 4 (Mut 4), and Mutation 5 (Mut 5) — structures deemed the most stable and closest in similarity to the control structure following a preliminary 100 nanosecond (ns) long MD simulation — were selected for further examination.

Table 1

Mutant pediocin constructs selected for thermostability analysis
Mutation	Region of Mutation
Mut 1	Ser13→Cys13 Gln39→Cys39
Mut 2	His38→Cys38 Gly40→Cys40
Mut 3	Asp17→Cys17 Lys20→Cys20
Mut 4	Thr35→Cys35 Gly37→Cys37
Mut 5	Asn28→Cys28 Gln39→Cys39

It is noteworthy that structures displaying inadequate stabilization during the pre-screening phase were excluded from the study. Figure 2 illustrates the results of RMSD, RMSF, and DSSP analyses conducted during the 100 ns long MD simulation.

The introduced mutations are expected to exert significant influences on both the structural and functional attributes of the protein. This anticipation arises from the recognition that each amino acid possesses distinct properties related to charge, size, polarity, and hydrophobicity. To discern the potential effects of these mutations on both structure and function, a comprehensive analysis of the respective mutants was conducted utilizing the HOPE server [21]. The summarized results are presented in Table 2. Notably, among the mutations implemented, only Mut 4 exhibited an amino acid substitution resulting in a change in polarity. However, this specific mutation, particularly when assessed in the context of SASA (Solvent Accessible Surface Area) values (as illustrated in Fig. 3A and 3B), did not give rise to any discernible changes. In these mutations, the reference peptide was replaced with Cys amino acids, known for their more hydrophobic nature compared to the amino acids originally occupying the same positions within the peptide sequence. Although this substitution was initially thought to confer added stability to the structure, a closer examination reveals that the Thr35→Cys35 mutation, particularly in Mut 4, leads to a 3% reduction in hydrophobicity at 373 K (100°C) when compared to the reference structure. Furthermore, this mutation results in a loss of hydrophobicity in the neighboring amino acids. Similarly, Mut 5 experiences a 2% decrease in hydrophobicity at 394 K (121°C) due to the Gln39→Cys39 mutation when compared to the reference peptide. These findings collectively suggest that the diminished hydrophobicity within the helical region, responsible for antimicrobial activity, leads to an expansion in the solvent-accessible area and subsequently renders the structure more susceptible to denaturation.

Table 2

Possible effects of changing amino acids on the structure and function of the peptide
Mutation	Region	Polarity	Charge	Hydrophobicity	Size of Residue
Mut 1	S13C	Polar/ Not changed	Neutral/ Not changed	More Hydrophobic	Not changed
Mut 1	Q39C	Polar/ Not changed	Neutral/ Not changed	More Hydrophobic	Decreased
Mut 4	T35C	Polar/ Not changed	Neutral/ Not changed	More Hydrophobic	Decreased
Mut 4	G37C	Apolar →Polar	Neutral/ Not changed	More Hydrophobic	Increased
Mut 5	N28C	Polar/ Not changed	Neutral/ Not changed	More Hydrophobic	Decreased
Mut 5	Q39C	Polar/ Not changed	Neutral/ Not changed	More Hydrophobic	Decreased

Upon interpreting the changes in SASA based on residues in both the reference and mutant peptides across varying temperatures, it becomes evident that hydrophobicity remains remarkably consistent in all mutants within the temperature range of 313–323 K (40–50°C). This consistency is particularly prominent within the "TTCIINNGALAWA" residues, which constitute the hydrophobic region of the peptide.At 313 K (40°C), the most structurally stable configurations are observed in the following order: the reference peptide, Mut 4, Mut 5, and Mut 1, respectively. A similar pattern is observed at 323 K (50°C). However, when assessing high temperatures (373–394 K or 100–121°C), the data from SASA graphs (Fig. 3) highlight that Mut 4, following the reference peptide, demonstrates the highest degree of stabilization. The minimal fluctuations observed in the SASA graphs for Mut 4, in conjunction with these changes closely mirroring those in the reference peptide's structure, particularly at 394 K (121°C) concerning the crucial antimicrobial site, strongly suggest that this mutant may exhibit resistance in various industrial processes.

Glycine stands out as a notably more flexible amino acid residue compared to all other amino acid types. This heightened flexibility endows it with a pivotal role in shaping the structural conformation of proteins, particularly in the context of torsional angles [21]. However, when it comes to the Gly37→Cy37 amino acid substitution in Mut 4, it's reasonable to anticipate a loss in this inherent flexibility. Remarkably, MD analysis revealed that this specific mutation, Gly37→Cy37, actually enhances the stability of the helical structure, particularly at elevated temperatures. This outcome suggests that the mutation may have a stabilizing effect on the protein's structure, offsetting the expected reduction in flexibility associated with Glycine. Another noteworthy amino acid substitution is Asn28→Cys28, and it's essential to recognize that this mutation occurs within the hydrophobic region. Asparagine is inherently less hydrophobic when compared to Cysteine. On this basis, one might initially expect the Asn28→Cys28 mutation in Mut 5 to promote greater stability within the hydrophobic region. However, somewhat surprisingly, the results from the simulations indicate that Mut 5 exhibits the least stability among the mutants. This outcome suggests that the interplay between amino acid properties and the broader structural context can yield unexpected effects, highlighting the intricate nature of protein behavior.

Evaluation of MD Simulations in Terms of RMSD, RMSF, and DSSP

While Pediocin PA-1 has traditionally found application in the food industry, where it serves as a safeguard against pathogen contamination in fermented meat products, recent research suggests its potential utility in the milk and dairy products sector. Its stability in aqueous solutions, wide tolerance for pH variations, and minimal conformational changes when subjected to heating and freezing processes position pediocin as a versatile bacteriocin candidate with promising applications across the food industry [31–33]. In addition to its intended role in food preservation, there have been reports of pediocin's potential in cancer treatment [34]. According to studies in the scientific literature, it is believed that the hydrophobic region of the peptide could play a pivotal role in generating anti-carcinogenic effects. This is attributed to the hypothesis that increased hydrophobicity enhances the peptide's interaction with mammalian cell membranes, potentially leading to cytotoxic effects [35, 36].

MD simulations were conducted for both the reference molecule, Pediocin PA-1, and the mutant peptide molecules across a spectrum of temperatures, spanning from 298 to 394 K (equivalent to 25 to 121°C). These simulations were extended over a duration of 300 ns. The evaluation of RMSD was employed to assess the stability alterations within these molecules throughout the simulation process.

RMSD is an effective metric for gauging the extent of fluctuations exhibited by atoms within proteins concerning the reference structure. This metric proves especially advantageous in the context of thermostability assessments as it provides insights into the structural conformational changes experienced by macromolecules.

Class IIa bacteriocins, which include pediocin-producing variants, are renowned for their remarkable thermal stability, enduring temperatures as high as 121°C (11). To assess the thermal stability of both the original and mutant peptides within the temperature range of 313 K to 323 K (40°C – 50°C), the data derived from the MD simulations were depicted using RMSD plots (Fig. 4). Figure 4A provides insight into the thermal stability of the reference and mutant peptide structures at 313 K (40°C). Notably, the original molecule and the Mut 4 peptide structure exhibit analogous deviations throughout the duration of the MD simulations. Specifically, it is apparent that all peptide structures display similar deviations, typically falling within the range of about 0.1 to 0.4 nm, for the initial 85 ns of the simulation. This consistency suggests that, up to this point, the structures remained remarkably stable, akin to the reference structure at 313 K (40°C) [37]. Concurrently, the SASA data acquired corroborate this assessment by characterizing Mut 4 as a stable mutant (Fig. 3A and 3B). However, a notable deviation emerges with the Mut 1 peptide, which experiences a swift increase from 0.4 nm to 0.6 nm after the initial 85 ns. Subsequently, it proceeds to exhibit a progressively aberrant profile, peaking at around 0.8 nm for the remainder of the simulation. This erratic behavior indicates a substantial loss of structural stabilization at 313 K (40°C). Furthermore, another peptide displaying a rapid deviation at 130 ns at 313 K (40°C), followed by irregular fluctuations in its structure throughout the simulation, is identified as Mut 5. This dynamic suggests a diminished stability profile for this mutant under the specified conditions.

Figure 4B illustrates the thermal stability of both the reference and mutant peptides at 323 K (50°C). During the initial 25 ns, all structures exhibit comparable deviations. Although, for the first 50 ns, the reference, Mut 4, and Mut 5 structures display parallel deviations, the distinctive fluctuation profile exhibited by Mut 4, particularly in the 75–150 ns range, stands out as noteworthy. This pattern implies that, at this temperature, Mut 4 exhibits relatively stable structural fluctuations, with a tendency towards consistency. This suggests that Mut 4 may experience reduced structural fluctuations and enhanced stabilization at this temperature.

However, despite the fluctuations observed at 313 K (40°C), all structures, especially at 323 K (50°C), maintain deviations within the range of 0.4–0.8 throughout the entirety of the simulation. This indicates that, at this higher temperature, all structures retain a relatively higher degree of stability, a crucial characteristic for peptides to preserve their antimicrobial activity.

The analysis of RMSF and DSSP provides additional support for the investigation into thermal stability conducted through MD simulations. When examining the RMSF results of the simulations performed at both 313 K (40°C) and 323 K (50°C) (Fig. 5A-B), it becomes evident that Mut 1 and Mut 5 display pronounced fluctuations at 313 K (40°C), particularly within the initial ten amino acid sequences (KYYGNGVTCG). This observation aligns with the RMSD and DSSP values. In accordance with the DSSP results, it is discerned that, at 313 K (40°C), Mut 1 experiences heightened deviations in regions associated with the hydrophobic helical structure, known for its antimicrobial effects, and a well-protected area inclusive of the "-sheet" (Fig. 5C-a). Additionally, the non-stable helical structure is prominently observed, further corroborated by SASA graphics. It becomes apparent that the α-helix structure cannot be maintained at this temperature and instead transitions into a 310-helix structure. Given that the 310-helix structure inherently represents an unstable helical conformation [38], this shift adversely impacts the stabilization of Mut 1, specifically within the peptide's active site, under these conditions. The RMSF and DSSP data collectively indicate that the active region of Mut 1 is not effectively shielded at 323 K (50°C). Toward the end of the simulation, a secondary structure resembling a "-sheet" structure emerges within the region previously occupied by the helical structure.

Similarly, Mut 5 exhibits a loss of its α-helix structure toward the latter part of the simulation, transitioning into a secondary structure profile primarily characterized by turn structures. The graph (Fig. 5) clearly illustrates that the substitution of Asn28 with Cys28 in Mut 5 triggers significant structural fluctuations commencing from Gly29, thereby contributing to destabilization within the peptide. It is reasonable to infer that Mut 1 and Mut 5 do not effectively preserve their helical structures when compared to the original peptide form. Consequently, their antimicrobial activities are expected to be notably diminished at both 313 K (40°C) and 323 K (50°C). Conversely, the helical regions within Mut 4 are presumed to remain relatively stable at 313 K (40°C) when compared to the control, suggesting that its antimicrobial activity may approach or even match that of the reference peptide. However, this scenario is reversed for Mut 4 at 323 K (50°C), where it exhibits a considerably less stable structural profile. The transformation of Gly37 to Cys37 renders Mut 4 more stable than the reference peptide structure, a conclusion supported by the SASA values derived from the simulations.

To assess the antimicrobial potential of pediocin, especially in the context of the dairy industry, simulations were conducted at elevated temperatures, a scenario that it often encounters during sterilization processes and has been demonstrated to retain activity in various studies [39, 40]. Figure 6 presents the RMSD data for the reference and mutant peptide structures at 373 K (100°C) and 394 K (121°C). Notably, the deviations observed in the peptide structures, typically within the range of 0.1–0.8 nm at 313 K (40°C) and 323 K (50°C), extend to the range of 0.2–1.25 nm at 373 K (100°C). This indicates significantly reduced structural stabilization at these higher temperatures. In particular, Fig. 6A reveals that Mut 1 exhibits a tendency to stabilize within the 0.8-1.0 nm range after 10 ns, maintaining structural stability with the exception of a sharp fluctuation within the 120–125 ns range. Similarly, Mut 5, which initially demonstrates substantial fluctuations over the first 75 ns but subsequently settles within the 0.8-1.0 nm range, appears to maintain a relatively higher degree of stability at 373 K (100°C) when compared to the reference and Mut 4 peptides. Upon examining the deviations in peptide structures at 394 K (121°C), the highest temperature utilized in the MD simulation, it is observed that the structures tend to stabilize at levels ranging from 0.6–0.7 nm, beginning from 0.1 nm. Notably, while Mut 5 exhibits the most pronounced fluctuations at 394 K (121°C), the reference structure, Pediocin PA-1, demonstrates the most stable structure with the least deviation. Consequently, it is advisable to investigate the activity of Mut 4, Mut 1, and Mut 5, respectively, under in vitro conditions to provide experimental validation and further insight into their potential [40].

Figure 7 presents the RMSF and DSSP data obtained from MD simulations conducted at elevated temperatures. The RMSF values, which typically exhibited deviations within the range of 0.2–1 nm at 373 K (100°C), expanded to 0.4–1.1 nm at 394 K (121°C). At 373 K (100°C), the DSSP data indicates that the reference peptide (Fig. 7C-a) has undergone nearly complete denaturation and has lost its significant secondary structure. Similarly, while the Mut 4 peptide has experienced a loss of secondary structure at 373 K (100°C) (Fig. 7C-c), the -sheet and 310 helix structures, characterized by high deviations, were periodically observed in the structures of Mut 1 and Mut 5 throughout the simulation (Fig. 7C-b and d). However, these structures were not observed to be stable at 373 K (100°C) due to their elevated deviation rates. At 394 K (121°C), the DSSP data indicates that the reference structure exhibits a tendency to assume -sheet structures; however, the RMSF values still suggest a high degree of instability. This suggests that while the reference structure tends to adopt a fixed conformation, it remains inherently unstable at this temperature. Despite the known resistance of bacteriocin class IIa peptides to temperatures ranging from 100–121°C [11, 41], the relative instability of the pediocin PA-1 structure, the control structure, at 394 K (121°C) raises questions regarding its antimicrobial effectiveness at such elevated temperatures. Notably, Mut 4 is a peptide structure in which the secondary structure in the region responsible for antimicrobial activity appears inclined toward renaturation. Although a helical structure is momentarily observed in this region during the simulation, DSSP analyses predominantly indicate a tendency to form -sheet structures. Conversely, Mut 1 and Mut 5 fail to establish a clear secondary structure pattern at 394 K (121°C) due to excessive deviations in both RMSF and DSSP analyses. Nonetheless, residues predisposed to -sheet formation continue to be observed. Collectively, all data obtained at temperatures of 373 K (100°C) and 394 K (121°C) suggest that Mut 4 is a peptide that may undergo a more stable renaturation process following structural disruption when compared to the control structure. Consequently, it is advisable to investigate the in vitro antimicrobial effects of Mut 4.

Evaluation of MD Analysis in Terms of Disulfide Bonds and Antimicrobial Activity Potential

The mechanism through which pediocin exerts its antimicrobial activity involves a hydrophilic structure, where the -sheet region plays a critical role in integrating the hydrophobic α-helix structure responsible for antimicrobial activity. This integration acts as a hinge within the lipid layer of the plasma membrane and involves the hairpin-like tail at the C-terminal. Additionally, pediocin is thought to exhibit antimicrobial activity by interacting with the mannose phosphotransferase system [1]. In the case of the Ser13→Cys13 conversion in Mut 1, it is predicted that the Cys amino acid tends to reside within the helical region, potentially disrupting the stabilization of the -sheet region. Analysis of Mut 1 using DSSP data indicated an increase in the stabilization of the -sheet region at 298 K (25°C) compared to all other peptides. However, this mutation led to a substantial disruption in the stabilization of the -sheet structure at various temperatures, resulting in the formation of random coils. Given the pivotal role of the -sheet structure in integration, it was also observed that Mut 4 was the mutant most inclined to maintain overall structural stability, including the -sheet region, particularly at elevated temperatures.

Bedárd et al. [8] proposed that the stabilization of the second disulfide bond (Cys24-Cys44) and its potential to enhance heat resistance might be attributed to the robust 2.4 Å hydrogen bond formed between His12 and Gln39. It was observed that, at 298 K (25°C), mutations generally led to an increase in the bond length between these two residues. Notably, in the case of Mut 4 (Fig. 8c), these mutations contributed to preserving the helical structure and enhancing the stability of the mutant peptide at room temperature. As for Mut 1, the expansion of the bond between these two amino acids to 3.1 Å is believed to aid in safeguarding the -sheet structure, which is believed to be crucial for cellular integration at this temperature (Fig. 8b).

The effects of mutations and temperatures on disulfide bond lengths have been summarized in Table 3. When evaluating disulfide bonds based on the Cα-Cα bond length, which is typically between 3.5 Å and 7.5 Å (42), it's important to note that in the literature, the C-C bond length between two Cys residues is approximately 5.5 Å, and S-S bonds typically measure around 2.04 Å on average [43]. One of the study's aims was to investigate the potential formation of a third disulfide bond, but unfortunately, in silico simulations did not support the possibility of forming a third disulfide bond for pediocin and its mutant molecules under any conditions. At 313 K (40°C), there was no significant difference in the lengths of disulfide bonds between the reference structure and mutant peptides. This suggests that the mutants could remain stable and exhibit antimicrobial activity at least to the same extent as the reference peptide. However, at 323 K (50°C), Mut 1 lost its second disulfide bond during the simulation, indicating low structural stabilization. This raises doubts about its ability to display antimicrobial activity. At 373 K (100°C), the original molecule, Pediocin PA-1, also lost its second disulfide bond, suggesting reduced stability compared to other mutants. Therefore, its antimicrobial activity at this temperature should be examined in vitro. On the other hand, Mut 5 lost its secondary disulfide bond at 394 K (121°C), indicating that it is unlikely to exhibit antimicrobial activity at this high temperature. In contrast, Mut 4 did not lose its secondary disulfide bond at any of the temperatures tested. Given the role of the second disulfide bond in antimicrobial activity, it is estimated that Mut 4, which maintains its disulfide bond stability at various temperatures in MD simulations, may also exhibit antimicrobial activity in vitro conditions.

Table 3

Effect of different temperatures on disulfide bonds and lengths in reference and mutant peptides.
Temperature	Peptid (reference or Mutant)	Residues	Cα - Cα	Cβ - Cβ	S-S
313 K (40°C)	Control	Cys9-Cys14	3.8 Å	3.9 Å	2.0 Å
	Control	Cys24-Cys44	5.9 Å	3.5 Å	2.0 Å
	Mut1	Cys9-Cys14	3.9 Å	4.1 Å	2.1 Å
	Mut1	Cys24-Cys44	6.0 Å	3.5 Å	2.0 Å
	Mut4	Cys9-Cys14	4.0 Å	3.9 Å	2.0 Å
	Mut4	Cys24-Cys44	6.0 Å	3.8 Å	2.0 Å
	Mut5	Cys9-Cys14	4.0 Å	3.9 Å	2.1 Å
	Mut5	Cys24-Cys44	6.1 Å	3.7 Å	2.0 Å
323 K (50°C)	Control	Cys9-Cys14	4.1 Å	4.2 Å	2.1 Å
	Control	Cys24-Cys44	6.2 Å	3.8 Å	2.1 Å
	Mut1	Cys9-Cys14	4.1 Å	4.0 Å	2.0 Å
	Mut4	Cys9-Cys14	3.8 Å	3.8 Å	2.1 Å
	Mut4	Cys24-Cys44	6.1 Å	3.6 Å	2.0 Å
	Mut5	Cys9-Cys14	3.9 Å	4.0 Å	2.0 Å
	Mut5	Cys24-Cys44	6.0 Å	3.7 Å	2.0 Å
373 K (100°C)	Control	Cys9-Cys14	4.0 Å	4.0 Å	2.1 Å
	Mut1	Cys9-Cys14	3.9 Å	4.1 Å	2.0 Å
	Mut1	Cys24-Cys44	6.0 Å	3.8 Å	2.9 Å
	Mut4	Cys9-Cys14	4.0 Å	4.1 Å	2.1 Å
	Mut4	Cys24-Cys44	5.9 Å	3.5 Å	2.1 Å
	Mut5	Cys9-Cys14	4.2 Å	4.2 Å	2.0 Å
	Mut5	Cys24-Cys44	5.9 Å	3.6 Å	2.1 Å
394 K (121°C)	Control	Cys9-Cys14	4.0 Å	4.0 Å	2.1 Å
	Control	Cys24-Cys44	6.0 Å	3.7 Å	2.1 Å
	Mut1	Cys9-Cys14	3.8 Å	3.8 Å	2.1 Å
	Mut1	Cys24-Cys44	6.0 Å	3.6 Å	2.0 Å
	Mut4	Cys9-Cys14	4.1 Å	4.1 Å	2.0 Å
	Mut4	Cys24-Cys44	5.9 Å	3.9 Å	2.0 Å
	Mut5	Cys9-Cys14	4.1 Å	3.9 Å	2.0 Å

This study provides valuable insights into the thermal stability and structural changes of pediocin PA-1 and its mutant structures created through Cys mutations. It's interesting to note that the addition of Cys mutations did not result in the formation of a third disulfide bond in any of the structures, even though the study aimed to investigate this possibility. Additionally, it was observed that the reference pediocin PA-1 structure could lose its secondary disulfide structure at high temperatures (100°C), which raises questions about its stability under such conditions. The mutant peptide named Mut 4, on the other hand, demonstrated superior thermal stability, maintaining its disulfide bond structure even at high temperatures. This suggests that Mut 4 may have potential applications in industries that require the stability of antimicrobial peptides at elevated temperatures. It's essential to emphasize that these findings are based on in silico simulations, and further experiments in vitro will be necessary to validate the results and assess the practical applicability of Mut 4 in real-world scenarios. If supported by in vitro data, the study suggests that Mut 4 could serve as a more stable version of pediocin for industrial use, which could have significant implications in various sectors, including the food industry. Overall, this research represents a promising avenue for the development of thermally stable antimicrobial peptides, and it highlights the importance of combining computational simulations with experimental validation to advance our understanding and application of such peptides.

Acknowledgments All in silico processes in this research were carried out at TÜBİTAK ULAKBİM, High Performance and Grid Computing Center (TRUBA resources).

Author Contribution Conceptualization: EGA, BS. Analysis and data curation: BS, EGA. Funding acquisition: EGA. Methodology: BS, EGA. Investigation: BS. Supervision: EGA. Writing – original draft - review & editing: BS, EGA. All authors have read and agreed to the published version of the manuscript.

Funding This work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK), Grant No: TOVAG-119O343.

Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest The authors declare no competing interests.

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Molecular Dynamic Study on the Structure and Thermal Stability of Mutant Pediocin Pa-1 Designed to Contain Extra Cystein Residues

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Introduction

Materials and Methods

Obtaining the three-dimensional structure of Pediocin PA-1 and constructing mutant molecules

MD Simulations

Results and Discussion

Evaluation of MD Simulations in Terms of RMSD, RMSF, and DSSP

Evaluation of MD Analysis in Terms of Disulfide Bonds and Antimicrobial Activity Potential

Conclusion

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References

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