1. Introduction
The application of green and biocompatible protocols for the creation of biomaterials appears as a crucial priority in recent literature [
1]. Preparation of biomaterials using physical associations between biocompatible macromolecules opens a great variety of possibilities for the development of nanostructures for medical [
2], food [
3], and agricultural [
4] industries. Electrostatically driven protein/polyelectrolyte complexation was extensively studied in the last three decades [
5,
6] and led to promising biomaterials, including nanoparticles for drug delivery [
7,
8], bioadhesive scaffolds for articular cartilage regeneration [
9], and microcapsules for the entrapment and viability improvement of probiotic bacteria [
10]. Proteins along with their specific functions offer multifunctionality as they have hydropathy and pH-tunable charge surface distributions [
11]. Therefore, they can interact with electrically charged macromolecules and can encapsulate hydrophobic substances. Apart from the exploitation of self-association in such systems, the use of mild thermal treatments supports the development of new materials because biopolymers and especially proteins alter their conformations under temperature changes [
12]. A well-documented case is the one of bovine serum albumin (BSA), which opens its conformation at elevated temperature to expose hydrophobic regions, which drive inter-protein associations by intermolecular β-sheets upon cooling back to room temperature [
13]. This feature was used to stabilize chondroitin sulfate/BSA electrostatic complexes against disintegration at neutral pH [
14]. Similar effects were utilized for β-lactoglobulin [
15].
Lysozyme is often used as a model protein for studies of protein/polyelectrolyte complexes [
16]. These complexes can adopt various structures (gel, microgel, micelle, colloid, precipitate, and coacervate) by adjusting the lysozyme/polyelectrolyte ratio as well as the nature and length of the polyelectrolyte [
17,
18,
19,
20]. Additionally, it was found that the impact on the structure and activity of lysozyme was determined by its interactions with polysaccharides. After thermal treatment, κ-carrageenan and konjac glucomannan were found to improve the stability of lysozyme-based complex systems [
21]. FTIR spectroscopy revealed that κ-carrageenan induces the rise of β-structure in lysozyme [
22].
The role and nature of molecular interactions in protein/polyelectrolyte complexes [
23] is fundamental for designing and optimizing these materials. The effects of complexation and the temperature response on the protein conformation are open questions for understanding the structural arrangement of the building blocks in the complexes, their mode of interaction, the binding with other molecules and their response or tolerance to external stimuli. For this purpose, atomic information is crucial in order to shed light on the dominant interactions and the responsive protein sites. Simulations provide considerable insight into the structure and the behavior of complexes. The recognition of the polyelectrolyte–protein complex coacervate binding mechanism in terms of both conformational preferences and energetics is significant for fine tuning of suitable parameters towards the formation of complexes with specific properties for different applications.
Various computational approaches and models have been employed to address these important questions [
24,
25,
26,
27,
28,
29,
30]. A very recent work focused on the effect of hydrophobicity on the sequence-dependent peptide conformation in polyelectrolyte complexes using molecular dynamics (MD) simulations [
31]. The study revealed that the peptide conformation and degree of hydrogen bonding are influenced by the specific sequence of the peptides, showing sensible trends consistent with experimental results. The importance of sequence-dependent conformational properties and hydrogen bonding of proteins is highlighted in the work of Arnittali et al. as well [
32]. In another study by Xu et al. [
33], the interaction of proteins with polyelectrolytes was investigated using a combination of experimental work, simulations, and mean-field theories, where the important role of counterions was demonstrated [
34]. Moreover the replica exchange technique [
25] was utilized to explore the way that charged polymers affect the thermal stability of beta-hairpin peptides. Sofronova et al. [
30] employed atomistic MD simulations to explore the complexation of the cationic protein lysozyme with poly(styrene sulfonate) and polyphosphate of different degree of polymerization and highly charged polyanions. It was observed that the shorter charged chains bound almost completely with the protein, while longer chains exhibit unbound terminal segments that adopt charged loop and tail conformations. Furthermore, Monte Carlo simulations using a simple model were used to examine the complexation between a single protein with a single polyelectrolyte [
28,
29]. Electrostatic interactions were found to be the major factor in forming these complexes, especially when the protein is highly charged at low ionic strength conditions. However, the study also showed that electrostatic attraction is not the only force driving these complexes to form.
These studies highlight the importance and necessity of detailed analysis at the microscopic level to understand the origin of key interactions between proteins and polyelectrolytes and to determine sequence–property relationships. Fully atomistic simulations provide the most comprehensive approach to achieve this, enabling a thorough exploration of how individual atoms contribute to a molecule’s specific behavior.
The current work is based on MD simulations of an aqueous solution of the biocompatible macromolecules poly(acrylic acid) (PAA) polyelectrolyte and lysozyme [
35,
36]. Studying their association could be useful for designing bioadhesives, tissue engineering scaffolds, or other biomedical applications that rely on interactions between polymers and proteins [
37]. Furthermore, it is a simple model system for the understanding of fundamental interactions between charged polymers and proteins. This study delves into the atomic-level impact of PAA on lysozyme’s conformational properties and investigates the effects of temperature on the protein’s structure and potentially on its enzymatic activity. We comprehensively examine how a quenching process influences the conformations of both PAA chains and the protein within the complexes, which is connected to a biocompatible treatment that induces interprotein associations and enhances stability in protein-based systems. All-atom MD simulations with explicit solvent model and explicit ion representation are utilized. By analyzing the protein’s secondary structure and the relative positioning of the two molecules within the complex, we elucidate their structural arrangement (i.e., the recognition of polyelectrolyte–protein complex coacervate binding sites). Energetic interactions, including electrostatic, Van der Waals, and hydrogen bonding components, reveal the primary driving force for complex formation and quantify the binding affinity between PAA and lysozyme. All these measures highlight short-range conformational changes of the protein molecules and validate the stability of the complex and the temperature-dependent reversibility of the complexation process. This detailed microscopic characterization, which to the best of our knowledge is performed for the first time, provides a comprehensive understanding of the factors governing the system’s behavior under specific conditions. This approach is essential for designing and optimizing biomaterials with precisely controlled properties.
4. Experimental Evidence
FTIR is sensitive to specific bond vibrations associated with secondary structures and CD is sensitive to the chiral nature of protein structures. These techniques are typically used to determine protein conformational transitions [
5,
55]. However, the spectroscopic characterization of protein secondary structure is often partially unreliable when samples are not extremely pure and abundant [
56]. Therefore, these two methods are combined to provide a comprehensive view of the protein secondary structure under different solution conditions and upon interaction with other components [
57]. FTIR experiments were conducted to identify the conformation of lysozyme in the free state and in the complexes before and after thermal treatment.
According to Chang’s study [
58], native lysozyme remains completely stable at 328 K for up to 60 min. However, at a higher temperature of 338 K, approximately 55% of the native of lysozyme partially unfolds under similar conditions. The initial rate of lysozyme partial unfolding increases nearly 14-fold as the temperature rises from 338 K to 348 K. The analysis further indicates moderate structural changes of lysozyme within 20 min at a temperature of 348 K. In addition, Xu et al. [
21] reported that mild denaturation, self-aggregation, and phase separation occurred when the temperature exceeded 343 K. In this context, a thermal treatment temperature of 353 K was selected, which is slightly above the temperature that lysozyme partially unfolds. The amide I band (1700–1600 cm
−1) (
Figure 12a) was investigated as it contains information on the various structural components [
13]. The FTIR data were modeled using a superposition of Gaussian functions [
59,
60]. The deconvolution of the amide I signal required the use of seven terms (
Figure 12b). The optimal positions of the Gaussian peaks were assigned to the various structural components as detailed in
Table S2.
Table S3 presents the results of the estimation of the secondary structure of native lysozyme at pH around 7 from various previous studies [
61,
62,
63,
64,
65]. The contribution of each conformation appears to vary slightly across different studies as there are small differences in the selected assignments. Our results show very good agreement with the recent study of Sadat et al. [
62], where the same assignments with our study were used.
Therefore, the values 47% for α-helix, 18% for β-sheet, and 35% for β-turn (
Figure 13) for lysozyme in the free state without thermal treatment are considered reliable as a starting point for investigation in the several conditions of this study.
In lysozyme, there is an increase in absorption intensity at wavenumbers ~1600–1630 cm
−1 after thermal treatment indicating the formation of additional β-sheet structures (
Figure 12a). Indeed, there is an increase in β-sheet to 24%, which is accompanied by a decrease in α-helix to 22% (
Figure 13). β-turn is found to increase to 54%. These findings suggest the irreversible partial unfolding of lysozyme globular conformation to a more open structure with the possible formation of intermolecular β-sheets. The complexation of lysozyme with PAA results in a decrease in α-helix to 25 and 26% and an increase in β-sheet to 27 and 21% for r
m 0.01 and 0.03, respectively. Regarding the FTIR signal, the addition of PAA shifts the peak to slightly lower wavenumbers, indicating an increase in β-sheet and a decrease in α-helix content. There is a decrease in β-turn to 30 and 33%; however, most of the α-helix loss leads to the appearance of random coil with contribution 18 and 20% for r
m 0.01 and 0.03, respectively. It is evident that the interaction of lysozyme with PAA leads to a partial disruption of the helices towards a random conformation. Upon thermal treatment, there is a marked increase in the FTIR absorbance in the region corresponding to β-sheet structures. A-Helix decreases to 22 and 18%, β-sheet increases to 33 and 40%, and β-turn decreases to 27 and 25% for r
m 0.01 and 0.03, respectively. Random coil remains the same for r
m 0.01 and decreases to 17% for r
m 0.03. This shows that β-sheet formation is induced by thermal treatment also within the complexes. This effect was used in the past from members of our group for the stabilization of protein/polysaccharide NPs [
47,
66,
67].
According to the CD experiments, in the secondary structure of lysozyme in the native state α-helix (29%) is much higher than β-sheet (9%), which is in qualitative agreement with FTIR analysis (
Figure 13). Random conformation (which includes β-turns) has a percentage of 62%. Representative fitting curves in comparison with experimental data can be found in
Figure S5. The secondary structure as it is observed in CD undergoes a slight change after thermal treatment (
Figure 14). The addition of a small amount of PAA (r
m = 0.01) does not seem to significantly affect the protein structure (
Figure 14a). However, after thermal treatment a minor difference in the protein structure is observed between r
m = 0 and r
m = 0.01. When the amount of PAA increased further (r
m = 0.03), a remarkable change in the spectrum and consequently in the protein conformation is evident, which is more pronounced in the thermally treated sample (
Figure 14b). Thus, for free lysozyme, thermal treatment results in a decrease in the α-helix structure from 29 to 27 and an accompanying increase in the β-sheet structure from 9 to 14. The addition of PAA results in a decrease in a-helix to 13% for the high r
m (although this decrease is not observed for the low r
m) and a simultaneous increase in β-sheet to 11 and 27% for r
m 0.01 and 0.03, respectively. In thermally treated complexes α-helix dropped to 17% for r
m 0.01 and was diminished for r
m 0.03 while β-sheet was at 25 and 45% for r
m 0.01 and 0.03, respectively. It is evident that the results from CD support the ones of FTIR as they show clearly the main effects of partial protein unfolding because of thermal treatment and complexation, which are the decrease in α-helix and the increase in β-sheet conformation.
5. Linking MD Insights to Experimental Data and Broader Scientific Impact
Molecular simulations provide a finer classification of protein structures compared to experimental techniques. Therefore, eight different types of structures emerge from the secondary structure analysis (
Figure 3 and
Table 2), with some of them seldom detectable (i.e., π-helix, 3
10-helix) in experiments. On the contrary, FTIR [
68] and CD [
69] spectroscopies mainly assign the α-helical structures, while other helical structures are characterized as “random coil” or “others”. Therefore, the comparison between the two approaches is mostly qualitative rather than quantitative.
There appears to be a rough agreement in the initial percentages of the various structures, making an assumption to add up the different types of helices, found with DSSP analysis, in one (38.8%), and comparing with the a-helix detected experimentally, and the same for β-sheet added to β-bridge (10.4%), whereas for turn the percentage is (25.5%). Note here that the classification is based on the 129 residues of the model protein, which corresponds to the 100% of the protein structure. Coil and bend are two additional structures detected in the model system but not in the experiment, which can be responsible for any remaining differences. According to the above assumption an increasing order of model detected structures is as follows: “sum of all the remaining structures” > α-helix > β-sheet, in line with both FTIR and CD measurements. Concerning the effect of thermal treatment on the complex, simulations show a small decrease in the helices to 37.6%, but no change is detected to “β-sheet and β-bridge” conformations, at the specific cooling rate. However, changes exist on each individual structure as depicted in
Table 2, resulting in local rearrangements of protein, which explain in a detailed manner the partial change in structure observed experimentally.
Furthermore, the MD calculations can be used for the development and optimization of new materials. The knowledge of the number and the kind of amino acids that take part in attachments with the polyelectrolyte give a very clear picture of the ability of the protein globules to connect with the polyelectrolyte to create nanoclusters and 3D nano or macroscopic networks. It also allows for knowing and designing which amino acids are left for interactions with other compounds, e.g., other proteins, drugs, and nutrients. The information on the conformational changes in the protein from its native state can be used to optimize the desirable biomaterial, according to the needs of the application, in terms of functionality versus number of bonds and network structure. Similarly, the intensity of the thermal treatment could be potentially tuned to balance between the enhancement of structural stability of the nanostructures and the compromise of the functionality of the protein which is related to its native state.
6. Conclusions
We use all-atom MD simulations combined with experimental techniques to investigate the complexation between PAA and lysozyme at the atomic level. The focus is on the effect of PAA and temperature on the conformational properties of lysozyme. In addition, a specific thermal process (relevant to stabilization of the complexes with non-electrostatic bonds) is explored for the way that it influences the interactions and the complexation of these molecules in water. Our analysis reveals subtle changes in the secondary structure of lysozyme, suggesting a high degree of thermal stability. While most structural variations fall within the range of statistical uncertainty, some structural elements appear to undergo a partially irreversible change after the thermal treatment, in qualitative agreement with experimental findings where a temperature-induced, partially irreversible transition of lysozyme’s globular conformation towards a more open structure is observed. Atomic details relating conformational changes to energetic contributions and hydrogen bonding highlight the origin of partial protein unfolding.
By analyzing the protein’s structure and the proximity of the molecules within the complex, we can determine their specific arrangement and how they interact. Specific recognition sites where the polyelectrolyte binds to the protein are revealed based on a detailed analysis for the amino acids with the shortest approach to polymer chains at 298 K, 368 K, and 298 K after quenching. The major findings are the following: (a) Compared to other amino acids, arginine (ARG) shows a remarkably high and temperature-independent association rate with polymer chains. (b) At higher temperatures (368 K), tyrosine (TYR) shows a significant decrease in its tendency to approach the polymer compared to 298 K, while serine (SER) shows the opposite trend. This change is irreversible upon cooling back to 298 K; (c) lysine (LYS) shows a small increase in its approach towards the polymer at higher temperatures. This increase is reversible, returning nearly to its initial value after quenching. (d) Quenching the system reduces the frequency of asparagine (ASN) approaching the polymer chains.
Energy and hydrogen bond calculations highlight the driving forces of the aforementioned behavior. Interestingly, the number of hydrogen bonds between the protein and polymer stays the same regardless of temperature or thermal process. Despite the minimal impact on global conformation, temperature-induced rearrangements lead to localized variations in hydrogen bonding between specific amino acids and polymer. This aligns with the observations regarding the proximity between lysozyme’s amino acids and PAA. The enhanced hydrogen bonding which is observed between ARG and PAA solidifies ARG’s role as a stable binding point for lysozyme on PAA.
The affinity between the two molecules is quantified through the calculation of both electrostatic and van der Waals interactions. The major role is played by electrostatic interactions, which are stronger than the weak van der Waals between all pairs. The strength of attractions between the protein and polymer (both Coulombic and Lennard Jones) fluctuates with temperature, but there is no clear trend overall. PAA is most affected by temperature where electrostatic attraction between PAA chains weakens with temperature increase, whereas van der Waals attraction strengthens and this change is irreversible upon cooling.
Experimental results from circular dichroism (CD) for lysozyme and its complexes with PAA align well with the Fourier transform infrared spectroscopy (FTIR) data. Both techniques reveal a decrease in alpha-helix content and an increase in beta-sheet structure, which suggests that the thermal treatment and complexation process leads to partial unfolding of the protein.
Beyond explaining the protein’s conformational changes under these specific conditions, the atomistic detail, revealed by the MD simulations, provides invaluable guidance for future experiments. This computational analysis offers a framework for interpreting existing or new spectroscopic data on protein conformational changes observed in other polyelectrolyte/protein systems and under analogous thermal treatments.