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. 2024 Apr 3;25(5):2838–2851. doi: 10.1021/acs.biomac.4c00002

Effects of Charge Sequence Pattern and Lysine-to-Arginine Substitution on the Structural Stability of Bioinspired Polyampholytes

Jelena Dinic †,‡, Matthew V Tirrell †,‡,*
PMCID: PMC11094733  PMID: 38567844

Abstract

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A comprehensive study focusing on the combined influence of the charge sequence pattern and the type of positively charged amino acids on the formation of secondary structures in sequence-specific polyampholytes is presented. The sequences of interest consisting exclusively of ionizable amino acids (lysine, K; arginine, R; and glutamic acid, E) are (EKEK)5, (EKKE)5, (ERER)5, (ERRE)5, and (EKER)5. The stability of the secondary structure was examined at three pH values in the presence of urea and NaCl. The results presented here underscore the combined prominent effects of the charge sequence pattern and the type of positively charged monomers on secondary structure formation. Additionally, (ERRE)5 readily aggregated across a wide range of pH. In contrast, sequences with the same charge pattern, (EKKE)5, as well as the sequences with the equivalent amino acid content, (ERER)5, exhibited no aggregate formation under equivalent pH and concentration conditions.

Introduction

Polyampholytes represent a class of macromolecules containing both positively and negatively charged monomers. They are defined in part by the fraction of charged monomers, denoted as F = f+ + f– and charge asymmetry, δf = |f+ – f–|, where f+ and f– are the fractions of positively and negatively charged monomers, respectively. There has been a growing research interest in annealed polyampholytes, which are composed of monomers in which the degree of ionization depends upon pH (in contrast to quenched polyampholytes that have pH-independent ionization).1 The overall charge of an annealed polyampholyte is affected by variations in pH, leading to pH-dependent macromolecular properties (i.e., chain conformation2). Positively charged amino acids, lysine (Lys or K) and arginine (Arg or R), along with negatively charged glutamic acid (Glu or E) and aspartic acid (Asp or D), form a class of ionizable monomers that contribute to the pH-dependent overall charge of a polypeptide macromolecule. Polyampholytes with different sequences of positive and negative charges along a backbone have been synthesized and explored across diverse applications. These applications include designing membranes with enhanced antifouling properties,3−5 micellar self-assembly for drug delivery,6 and the investigation of conformation, secondary structures,7,8 and phase separation behavior9,10 in different classes of proteins. Ionizable amino acids are uniquely useful in these studies, as they enable macromolecular synthesis with a precisely controlled charge sequence pattern through solid-phase peptide synthesis. These sequences enable inferences about the charge sequence effects in larger polypeptides and proteins.

The proteins of interest in the aforementioned studies encompass those with a high fraction of ionizable amino acids, such as intrinsically disordered proteins (IDPs)11−13 and single stable alpha helices (SAHs).14−16 IDPs are recognized for their absence of stable secondary and tertiary structures,17 resembling synthetic macromolecules in solutions.18 Macromolecules with a fraction of charged monomers, F = 1, charge asymmetry, δf = 0 (an equal number of positive and negative charges) have been employed as model systems for intrinsically disordered proteins (IDPs) in studies that specifically focus on the impact of ionizable amino acids and proteins’ charge sequence pattern on their conformation13,18−20 and phase separation behavior.21−26 Similar to IDPs, single stable alpha helices (SAHs) also contain a high fraction of charged monomers. They are characterized by a charge pattern that closely follows a sequence consisting of four negatively charged Glu monomers, succeeded by four positively charged monomers represented by a mixture of Lys and Arg (E4(K/R)4). However, unlike IDPs, SAHs are known for the formation of long, stable helical structures.15 Therefore, previously published studies suggest that a polyampholyte with a high fraction of charged monomers can exist in a disordered state, lacking a stable secondary structure, but it can also exhibit a high degree of helicity. This observation emphasizes the significance of identifying factors that could influence the formation of secondary structures in polyampholytes. Closely related to this, we identify two relevant questions here: the first is related to determining how a variation in charge sequence pattern influences secondary structure formation, and the second focuses on how the type of positively charged monomers (i.e., Lys vs. Arg) affects the formation of secondary structure.

The helicity is determined by both the configurational entropy loss upon helix formation and the enthalpic gain originating from backbone hydrogen bonds, backbone–side chain interactions, and side chain–side chain electrostatic interactions and hydrogen bonding.8,27 The interactions between oppositely charged amino acids, involving a combination of electrostatic interactions and hydrogen bonding, can stabilize an α-helical structure. Among the interactions, those between Glu at the ith position along the sequence and Lys placed 4 monomers away (at i + 4 position) have been identified as the most helix-stabilizing and are known as i, i + 4 interactions.28,29 The i, i + 3 interactions (between the ith Glu and Lys placed 3 monomers away) are also found to be helix-stabilizing, although less so than i, i + 4 interactions.28 On the other hand, when oppositely charged amino acids are positioned in closer proximity, the interactions of i, i + 2, and i, i + 1 between Glu and Lys have been observed to destabilize the helix, favoring a disordered conformation.28 Examining the relatively consistent charge sequence pattern in SAHs, (E4(K/R)4)n, it becomes evident that the number of potential (i, i + 4) interactions between oppositely charged monomers, contributing to helix stabilization, is maximized. It has also been demonstrated that, in addition to stabilizing an α-helix through interactions between oppositely charged side chains, large side chains, such as Arg, Lys, and to a lesser extent, Glu, offer shielding to the peptide backbone from the solvent.30 This additional shielding further contributes to stabilization of a helical configuration. Our recent publication focused on how changes in the charge sequence pattern impact the secondary structure formation of sequence-specific polyampholytes.31 The investigation specifically considered polyampholytes of longer chain lengths with charge patterns different from those found in SAHs. The sequences of interest were (EKEK)n and (EKKE)n, where n = 25. The study unveiled the significant influence of the charge sequence pattern on the secondary structure preferences of polyampholytes at physiological pH. Moreover, it demonstrated the potential to modulate the secondary structure of the two examined sequences through external stimuli, including pH adjustments, variations in ionic strength, and changes in the solvent dielectric constant.

The influence of substituting Lys with Arg in sequence-specific polyampholytes is another relevant aspect to consider when discussing factors affecting secondary structure formation. The Lys side chain consists of four methylene groups and a positively charged amino group. The Arg side chain contains the guanidinium group with a planar geometry, consisting of three amino groups bound to a carbon atom. Both amino acids are positively charged under physiological conditions. As intrinsically disordered regions/proteins (IDRs/IDPs) are enriched in polar and charged amino acids, both Arg and Lys are recognized as disordered-promoting amino acids.32,33 The wild-type IDPs and their mutants containing the Arg-to-Lys substitution have been compared regarding their propensity to participate in biological liquid–liquid phase separation (LLPS).9,34,35 Arg-to-Lys substitution in IDP sequences reduces the tendency of IDPs to undergo phase separation, despite both amino acids carrying a positive charge. This is because Arg has a stronger ability to participate in cation−π interactions with aromatic groups than Lys. Additionally, unlike Lys side chains, Arg side chains have the capability to participate in π–π interactions owing to the quasi-aromatic sp2-hybridized guanidinium group.36,37 Guanidinium acts as a hydrogen bond donor, with water interactions present only in the molecular plane. However, above and below the molecular plane, guanidinium exhibits dehydration.38 The important role of hydrophobicity of Arg in the LLPS has been demonstrated recently.37,39 In particular, the study by Hong et al.37 focused on contrasting the LLPS of Arg-rich proteins and Lys-rich polypeptides with oppositely charged hyaluronic acid. This study highlighted the differences between Lys and Arg by examining the interplay of electrostatic interactions and hydrophobicity in the absence of aromatic amino acids.

Both Lys and Arg are also known for their helical propensity.33 Multiple studies have shown that the interactions of both Lys and Arg with the oppositely charged Glu can stabilize an α-helical secondary structure.7,27,28,40 The studies, which focused on examining i, i + 4 and i, i + 3 types of interactions between Lys and Glu,28 as well as those between Arg and Glu,40 revealed a difference in the stability of an α-helix after making a Lys-to-Arg substitution in both types of interactions.27 Additionally, a recent study on SAHs revealed significant differences between Glu-Lys and Glu-Arg interactions and their distinct effects on secondary structure.7 This effect was demonstrated through molecular dynamics (MD) simulations and Protein Data Bank (PDB) investigations, revealing that Glu-Arg ion pairs are more dynamic than Glu-Lys pairs, forming faster and having shorter lifespans compared to Glu-Lys ion pairs. Glu-Arg ion pairs can accommodate more side chain rotamer conformations than Glu-Lys pairs. The results suggest an important entropic contribution to the stability of the formed α-helix. Conversely, Glu-Lys ion pairs utilize fewer rotamer combinations but have a larger favorable enthalpy contribution to the overall free energy of α-helical formation.7 Notably, the analysis showed a higher frequency of Glu-Arg (i, i + 3) ion pairs compared to Glu-Arg (i, i + 4), in contrast to the trend observed for Glu-Lys ion pairs.7 The observation that Arg and Glu ion pairs occur more frequently also implies their potential formation between different molecules, contributing to polyampholyte aggregation. The fact that the charge sequence pattern plays an important role in the formation of secondary structures, coupled with recent studies suggesting that Lys-to-Arg substitution may also contribute, inspired us to conduct the investigation discussed here.

In this publication, we investigate the combined effects of the charge sequence pattern and the type of positively charged amino acids on the formation of secondary structure in sequence-specific polyampholytes. The charge sequence patterns of interest, EKEK and EKKE, differ from those found in SAHs. The amino acid substitution carried out in this study involves replacing Lys with Arg. Similar charge patterns, such as the ones examined here, have been previously studied in the context of IDPs, primarily through theoretical models and simulations.18,41 However, to the best of our knowledge, no experimental investigation has been performed to assess information regarding the potential tendency of these sequences to form a secondary structure, with a particular emphasis on amino acid substitution. Additionally, we investigated the stability of the formed secondary structures by introducing urea and NaCl. Urea is known for destabilizing secondary structures,42,43 and studying these polypeptides in the presence of urea at varying concentrations provides a useful means of comparing the stabilities of sequences with different charge patterns and types of monomers. On the other hand, NaCl is found to have both stabilizing and destabilizing effects on the secondary structure, depending on the concentration of NaCl and the polypeptide sequence.44 Since these sequences are composed of all ionizable monomers, the addition of NaCl has a pronounced effect on electrostatic interactions and, consequently, the stability of the formed structures. Lastly, we explore the possibility that these sequences form aggregates.

Materials and Methods

Sequence-Specific Polypeptides

Sequence-specific polypeptides of interest here are (EKEK)5, (EKKE)5, (ERER)5, (ERRE)5, and (EKER)5. E represents glutamic acid (also denoted as Glu), K represents lysine (Lys), and R represents arginine (Arg). The charge sequence patterns of interest and the corresponding amino acid structures are shown in Figure 1. The custom polypeptides were purchased from GenScript, Piscataway, NJ. Both N-terminal modification (acetylation) and C-terminal modification (amidation) were performed on these sequences. Polypeptide identities were confirmed using reversed phase high-performance liquid chromatography (RP-HPLC) (Figures S1–S5). Additionally, the electrospray ionization mass spectrometry (ESI-MS) spectra are shown in Figures S6–S10.

Figure 1.

Figure 1

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Polypeptide sequences used in the study and chemical structures of Lys, Glu, and Arg. The sequences examined here are (EKEK)5, (EKKE)5, (ERER)5, (ERRE)5, and (EKER)5.

Solution Preparation

Sequence-specific polypeptides were dissolved in Milli-Q water at room temperature. The concentration of the stock solutions was 1.0 mM, and all subsequent dilutions were performed in Milli-Q water. All as-prepared solutions are acidic (pH ∼ 3–4) due to the use of strong acid (TFA) during the peptide cleavage step and readily dissolve in water, except for (ERRE)5, for which the solution turns turbid upon mixing with water at a pH close to or above 3.5. The pH was adjusted to the desired pH using solutions of NaOH and HCl. pH adjustment was performed after diluting the stock solutions.

The (ERRE)5 polypeptides followed a slightly different preparation protocol compared with the rest of the polypeptide sequences. This adjustment was necessary due to the observed aggregation at 1.0 mM at pH ∼ 3.5–4, which persisted even upon dilution. Consequently, the polypeptides were initially prepared under more acidic conditions (pH below 3.0), and the dilution was carried out after this initial pH adjustment to a low pH. The pH was then adjusted to the desired value after the samples were diluted to a concentration near the target of 100 μM. By following this approach, the aggregation of (ERRE)5 was avoided, and successful dilutions were achieved, followed by pH adjustment to a desired pH value.

Circular Dichroism (CD) Experiments

Circular dichroism experiments were performed on a CD spectrometer (Jasco J-815 CD Spectrometer, Jasco Inc., Tokyo, Japan). CD spectra were collected between 260 and 190 nm in a quartz 1.0 mm cuvette. The instrument was set to record three spectra of each sample and provide the resulting average spectrum. Hence, each CD spectrum shown in this study represents an average of three consecutive scans. A constant scanning speed of 50 nm/min with a data pitch of 0.1 nm was used in all experiments. All experiments were performed at room temperature and at a concentration of 100 μM. The equations describing ellipticity and the necessary unit conversions are presented in Section S1. The primary reason that we cannot use methods that require fitting the entire range of wavelengths (usually in the 190–250 nm range) to determine secondary structure elements of solutions is that urea and NaCl solutions studied here exhibit high absorbance at wavelengths of around 205 nm and below. Ellipticity measured below 205 nm introduces significant noise, making it difficult to determine the secondary structure elements. However, this is not a concern in the present publication, where our focus is primarily on the analysis and discussion of the helical fraction of sequence-specific polypeptides. Therefore, we calculate the helical fraction, fH using a simple formula of the form8

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Here, [θ]222 represents experimentally determined ellipticity at 222 nm, and n represents the number of peptide bonds.8,45 [θ]coil represents the ellipticity of the disordered state: [θ]coil = 640 – 45T. T represents the temperature.

Turbidity Measurements

Turbidity experiments were performed on a Tecan Infinite M200 Pro plate reader with a UV spectrophotometer. A 96-well black plate with a clear bottom (BD Falcon) was used. The measurements presented here were taken at 450 nm. The sample measurements in the range of 350–650 nm are also shown in Figure S11. All experiments were conducted at room temperature and were repeated three times.

Results and Discussion

pH-Dependent Circular Dichroism (CD) Spectra of Sequence-Specific Polyampholytes

The CD spectra for five sequence-specific polyampholytes are displayed in Figure 2 at three distinct pH values. Figure 2a,b shows the CD spectra of two Lys-rich sequences, (EKEK)5 and (EKKE)5. The CD spectrum of (EKEK)5 at pH 3.0 is characterized by two minima at 222.4 and 204.1 nm and a maximum at 190 nm (defined as the highest measured positive value of MRE in the tested wavelength range of 190–260 nm). At higher pH values, the minimum at the higher wavelength disappears, indicating a drop in helicity. The helical contents of (EKEK)5 confirming this observation are shown in Figure 2a, bottom. The CD spectra of (EKEK)5 polypeptides appear to have the highest helical content at low pH, and this helical content decreases as the pH increases, eventually reaching zero. These polypeptide sequences exhibit similar behavior to that described in our previous publication for longer sequences, (EKEK)25 and (EKKE)25.31 The helical content of polypeptides is expected to be dependent on chain length, with shorter chains displaying lower helical content.45 This is mostly due to the high entropic cost of initiating helix formation. The costly helix-initiating step can only be justified by the enthalpic gain from helix propagation through hydrogen bond formation.46,47 Consequently, the propensity to form helices decreases as the chain length decreases, indicating a lower enthalpic gain in shorter sequences. Therefore, it is not surprising that these sequences exhibit lower helical contents than those reported for (EKEK)25.31

Figure 2.

Figure 2

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pH-dependent CD spectra. Mean residue ellipticity (MRE) against wavelength in the 260–190 nm range and helical fractions (fH) are shown at three distinct pH values for (a) (EKEK)5, (b) (EKKE)5, (c) (ERER)5, (d) (ERRE)5, and (e) (EKER)5.

The CD spectra of the other Lys-rich sequence (EKKE)5 display little variation with the pH (Figure 2b). The helicity is equal to zero for all three pH values (Figure 2b, bottom). This is consistent with our previously published results for longer sequences.31 One of the crucial reasons why polyampholytes with an EKEK pattern can form helices, while those with EKKE cannot, is related to the interactions between oppositely charged amino acids dictated by the charge pattern and their impact on helicity, as discussed in Introduction. Specifically, when the spacings between Lys+ and Glu– are (i, i + 4) or (i, i + 3), the interactions between the oppositely charged amino acids stabilize the helix.28 Among these, (i, i + 4) spacing provides greater stabilization of an α-helix, resulting in higher helicity than the other spacing. On the other hand, the spacings (i, i + 2) and (i, i + 1) tend to destabilize an α-helix while favoring disordered conformation. The sequences studied here cannot form (i, i + 4) ion pairs due to those interactions being consistently charge repulsive. Instead, these sequences can only form (i, i + 3) ion pairs among those pairs that provide helical stabilization. In our previous study, we discussed these effects and their implications for the helical content of the two charge patterns. Here, we show the possible ion pairs for the EKEK and EKKE charge patterns in the Supporting Information (Figure S12) and refer the reader to the publication31 for more details.

The CD spectra of (ERER)5 at three pH values are shown in Figure 2c, and the corresponding helical contents are displayed in Figure 2c, bottom. All three CD spectra exhibit two minima located near the helical minima at 222 and 208 nm. The calculated helical contents show modest dependence on pH, and the values are higher than any values calculated for other sequences. The (ERER)5 polypeptides demonstrate an unusual helical stability when compared to (EKEK)5 polypeptides with the same charge pattern.

The CD spectra of (ERRE)5 at three pH values (pH 3.0, 7.0, and 11.5) are shown in Figure 2d. The spectra exhibit little dependence on pH. The helical contents determined from these spectra reveal a consistent helical fraction of zero at all three pH levels (Figure 2d, bottom). The results for the (ERRE)5 system also confirm that the solution preparation method of dissolving polypeptides under very acidic conditions, followed by dilution and then adjusting pH to a desired level, represents a suitable way to avoid aggregation. These spectra and helical contents at all three pH values share significant similarities with those of (EKKE)5 (shown in Figure 2b). However, a distinct solution preparation method for (ERRE)5 polypeptides highlights the difference between these two systems, where the Arg-rich polypeptides encompassing this charge pattern exhibit a higher tendency to aggregate. The aggregation behavior of (ERRE)5 will be discussed later in this article.

The CD spectra of a sequence containing both Lys and Arg, (EKER)5, at three pH values, are shown in Figure 2e. Closer inspection reveals that these CD spectra exhibit features of both Lys-rich (shown in Figure 2a) and Arg-rich (Figure 2c). Similar to (ERER)5, the spectra of (EKER)5 display still modest but definitely stronger, pH-dependence. Additionally, the helical fractions of (EKER)5 (Figure 2e, bottom) are higher than those determined for (EKEK)5 (see Figure 2a, bottom). However, the calculated helical contents are lower than those calculated for (ERER)5, as shown in Figure 2e, bottom.

We also compare the experimentally determined helical fractions for the five sequences with those obtained using the helix–coil prediction algorithm, AGADIR48,49 (see Figure S13). For sequences with an alternating charge pattern, namely, (EKEK)5, (ERER)5, and (EKER)5, the algorithm predicts lower helical fractions at pH 3.0 than those determined experimentally. While values determined using eq 1 may overestimate helical fractions, a simple visual inspection of the spectra shown in Figure 2 reveals that experimentally achieved helical fractions are higher than those determined using AGADIR. For (EKEK)5, the AGADIR predictions at pH 7.0 and 11.5 are closer to the values determined experimentally. However, for the other two sequences, (ERER)5 and (EKER)5, the algorithm still predicts helical fractions that are lower than those determined experimentally in this study, with the highest discrepancy seen at pH 3.0. Interestingly, the algorithm predicts low helical fractions at both pH 3.0 and 11.5, confirming the unique occurrence of higher helical stability at low pH and the absence of helices at high pH observed experimentally, which the theory-based algorithm cannot capture. Lastly, the algorithm demonstrates a good agreement with experimentally obtained helical fractions (zero or close to zero) of (EKKE)5 and (ERRE)5 at all three studied pH.

In the following sections, our goal is to examine and contrast the stability of the secondary structures of Lys- and Arg-rich polyampholytes after the introduction of solute molecules. We do this by examining how the addition of urea and NaCl influences the stability of the helical structure. These solutes were selected for their capabilities to induce structural changes in proteins.50−52 NaCl is known for its salting-out capabilities53 and is known to promote the loss of secondary structure in short peptides where ionic interactions stabilize helical conformation.44,54 Urea is a potent protein denaturant.42 Monitoring changes in ellipticity at 222 nm (and, indirectly, helicity as shown in eq 1) by adding urea and NaCl is a means of assessing information about, and comparing the stability, of secondary structures in different peptide sequences. We divided the discussions on the effects of urea and NaCl on secondary structure formation into two groups. The first group includes sequences with the same alternating charge pattern: (EKEK)5, (ERER)5, and (EKER)5. The second group includes (EKKE)5 and (ERRE)5. Lastly, we present and discuss the aggregation behavior of (ERRE)5.

Secondary Structure Stability of (EKEK)5, (ERER)5, and (EKER)5 in Urea Solutions

Mean residue ellipticity (MRE) at 222 nm plotted against the concertation of added urea for (EKEK)5, (ERER)5, and (EKER)5 is shown in Figure 3. Figure 3a shows the urea concentration dependent MRE curves of (EKEK)5 at three pH values. All three curves show an increase in MRE with an increasing urea concentration, indicating a progressive loss of the secondary structure. Additionally, all three curves converge at high urea concentrations, suggesting that a similar degree of secondary structure disruption was reached for (EKEK)5 at three distinct pH values. The calculated helical fractions of (EKEK)5 at three pH values are shown in Figure 3b. A noticeable loss of helicity with an increasing urea concentration is observed only at pH 3.0. At pH 7.0, (EKEK)5 polypeptides without added urea exhibit a low helical fraction (7.5%), which vanishes completely at curea = 1.2 M. At pH 11.5, the helical content is equal to zero in the urea-free environment and remains zero at all concentrations of urea. While the present study primarily focuses on the analysis of the helical structure among the secondary structure elements, urea is a potent denaturant known for its ability to unfold proteins and disrupt their secondary structure. The fact that MRE of (EKEK)5 shown in Figure 3a becomes less negative with increasing urea concentration indicates that even when the helical content is equal to zero, there is still a loss of secondary structure consisting of structural elements other than α-helices, such as β-sheets and turns.

Figure 3.

Figure 3

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Mean residue ellipticity at 222 nm and helical fractions of (EKEK)5, (ERER)5, and (EKER)5 at three distinct pH values plotted against the concentration of urea. (a) MRE at 222 nm of (EKEK)5, (b) helical fractions of (EKEK)5, (c) MRE at 222 nm of (ERER)5, (d) helical fractions of (ERER)5, (e) MRE at 222 nm of (EKER)5, and (f) helical fractions of (EKER)5.

MRE of (ERER)5 polypeptides at three distinct pH values is shown in Figure 3c. (ERER)5 polypeptides demonstrate the capacity to maintain a helical fraction even at high urea concentrations, in contrast to the MRE curves determined for (EKEK)5 (shown in Figure 3a). This ability to maintain a helical fraction is demonstrated by (ERER)5 polypeptides at all three pH values, and it is especially pronounced at low pH, where the helical fraction remains the highest across all tested urea concentrations. To better illustrate the ability of Lys-to-Arg substitution to resist helical disruption, we plotted the urea-concentration-dependent MRE ratios of (ERER)5 and (EKEK)5, as well as the MRE ratios of (ERER)5 and (EKER)5 in Figure S14. The plots in Figure S14 demonstrate that the ratios continuously increase with urea concentration, implying a less potent effect of urea on disrupting the starting helical structure in the Arg-rich system. The results presented in this section reveal that the Lys-to-Arg substitution within the charge-alternating sequence leads to an enhanced helicity and resistance to helical disruption. Notably, both pH 3.0 and pH 7.0 correspond to the charged state of Arg, while at pH 11.5, Arg retains some of its charge, as it is close to the predicted pKa of Arg (pKa 12.5).55

The exact mechanism by which urea disrupts secondary structures may not be fully understood.56 Studies suggest that the presence of urea disrupts intrapeptide hydrogen bonds and alters the solvent environment, ultimately leading to the loss of the secondary structure.57 In the case of the Arg-rich sequence, experiments confirm high starting helicity at all three pH values (Figure 3d), and progressive loss of the helical fraction with increasing urea concentration. At pH 7.0, the enhanced stability of the helical structure is attributed to the ability of Arg side chains to form ion pairs with Glu monomers, as discussed earlier in the text. Studies suggest that the Arg side chain can potentially form an ion pair with two Glu side chains simultaneously, further contributing to the stability of the formed structures.7 However, at low pH, when Glu is uncharged, indicating an inability for ion pair formation, and at high pH, when Arg is only partially charged, a similar trend is observed. In fact, the greatest stabilization of the helical structure is seen at pH 3.0. Therefore, high helical fractions at all three pH values, irrespective of the charge state of side chains, suggest the helix-stabilizing role of hydrogen bonds formed between oppositely charged side chains. Urea is not potent in effectively destabilizing ion pairs formed between oppositely charged amino acids.58 Nevertheless, it should effectively disrupt both backbone hydrogen bonds and those formed between side chains, leading to loss of helicity.28 However, the fact that Arg-rich polypeptides maintain a higher helical fraction than Lys-rich polypeptides at the matched concentration of urea, along with the continuous increase in the ratios shown in Figure S14, suggests an additional mechanism of helical stabilization in Arg-rich polypeptides. This additional helical stabilization is possibly provided by the bulky Arg side chains, which offer greater shielding of the backbone from the solvent than the Lys side chains. The shielding ability of Arg, and bulky side chains in general, has been discussed in the past.30,59,60 Therefore, the effect of adding urea should be less potent than in the case of Lys-rich sequences at the matched urea concentration.

The urea-concentration-dependent values of MRE for the (EKER)5 sequence at three distinct pH values are shown in Figure 3e, and the corresponding helicity values are presented in Figure 3f. These polypeptides undergo a loss of secondary structure with added urea at all three tested pH values. The extent of this loss is similar to the loss experienced by (EKEK)5 (fH = 0 at the highest urea concentration tested), despite the fact that the initial (curea = 0 M) helicity values at each respective pH are higher than those of (EKEK)5. The helical contents (shown in Figure 3f) of this sequence are lower than those determined for (ERER)5. For (EKER)5, secondary structure disruption occurs at high urea concentrations (curea > 4.0 M). This indicates that the presence of Arg in (EKER)5 increases the initial helicity value compared to that in (EKEK)5, but the presence of Lys increases the overall extent of secondary structure disruption. The results presented here imply that the starting secondary structure as well as the extent of urea-induced disruption of the structure of the charge-alternating sequences strongly depend on the composition of the positively charged amino acids.

The helical fraction determined from eq 1 can be converted to the helix-propagation parameter s, and its dependence on urea concentration can be expressed. The helix-propagation parameter was introduced in the Zimm–Bragg model of the helix–coil transition.28,61,62 It describes how readily a protein forms a helical structure from a disordered state (intrinsic helix forming tendency). A higher s value indicates a greater propensity of a protein to form an α-helical structure. The discussion of the s parameter calculation procedure is included in the Supporting Information. The pH-dependent s values determined for (EKEK)5, (ERER)5, and (EKER)5 are shown in Figure S15a. The s values of (ERER)5 at all studied pH are higher than those of (EKEK)5 and (EKER)5. Additionally, the urea concentration dependent s values are shown in Figure S15b–d. At the matched urea concentration, (ERER)5 exhibits the highest values of s, while (EKEK)5 has the lowest calculated s value among the three sequences. The results indicate that it is easier for (ERER)5 to form a helical structure, and the formed structures remain more stable even at high urea concentrations compared to those of (EKEK)5 and (EKER)5.

Secondary Structure Stability of (EKKE)5 and (ERRE)5 in Urea Solutions

Mean residue ellipticity (MRE) at 222 nm of (EKKE)5 at three pH values is shown in Figure 4a. Unlike the charge-alternating polypeptides shown in Figure 3, the curves for (EKKE)5 show little pH dependence. The (EKKE)5 polypeptides do not exhibit helix formation at any of the studied pH values (Figure 4c), and the initial values of MRE are close to zero. Yet, the increase in MRE with urea concentration suggests, just as in the case of (EKEK)5 at pH 11.5, the loss of the secondary structure after the introduction of urea. As mentioned earlier, the loss of secondary structure in that case corresponds to the loss of other structural elements, including β-sheets and turns.63

Figure 4.

Figure 4

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Mean residue ellipticity at 222 nm and helical fractions of (EKKE)5 and (ERRE)5 plotted against the concentration of urea. (a) MRE at 222 nm of (EKKE)5 at three distinct pH values. (b) MRE at 222 nm of (ERRE)5 at pH 3.0. (c) Helical fractions of (EKKE)5 and (ERRE)5.

The urea-concentration-dependent MRE values of (ERRE)5 at pH 3.0 are presented in Figure 4b. As stated earlier, (ERRE)5 polypeptides tend to aggregate at pH levels above 3.0. Due to this aggregation behavior, pH adjustments can only be carried out at low concentrations (in this study, concentrations below 150 μM). As a result, we were unable to prepare samples with the desired range of urea concentrations at pH 7.0 and 11.5, because the polypeptides began to aggregate before the dilution using the urea solution could be performed. The MRE values, as shown in Figure 4b, increase with increasing urea concentration, following a similar trend to that of (EKKE)5. In addition, the helicity is consistently zero at all urea concentrations (Figure 4c), mirroring the situation observed in the (EKKE)5 system. The absence of a helical structure in both (EKKE)5 and (ERRE)5 at low pH indicates that substituting Lys with Arg does not necessarily increase helicity, contrary to what might be inferred from the results presented in Figure 3. In fact, the findings in Figures 3 and 4 strongly suggest that both charge sequence patterns and ionizable amino acid composition play roles in helical formation and the stability of formed helices. Altogether, the results for the loss of secondary structure with urea concentration reveal that Lys-to-Arg substitution may increase helical stability, but the effect is highly dependent on the charge sequence pattern. The results also suggest that the Arg-rich sequence, (ERER)5, retains some of its secondary structure at high urea concentrations independent of pH. In contrast, all other studied sequences have MRE values closer to zero, indicating a secondary structure considerably different from that observed for (ERER)5 at high urea concentrations. In the following section, we examine the structural changes exerted on sequence-specific polypeptides after the addition of NaCl. NaCl was introduced for its ability to disrupt electrostatic interactions between charged side chains.

Secondary Structure Stability of (EKEK)5, (ERER)5, and (EKER)5 in Salt Solutions

Mean residue ellipticity at 222 nm is plotted against the concentration of added NaCl at three distinct pH values for (EKEK)5, (ERER)5, and (EKER)5 in Figure 5. The MRE values of (EKEK)5 at three different pH levels are displayed in Figure 5a. The addition of NaCl has a minor effect on the MRE (and corresponding helicity) of (EKEK)5. At pH 3.0, there is a slight decrease in MRE, while at pH 7.0, a small increase in MRE is observed. At pH 11.5, the MRE values remain almost independent of the concentration of NaCl. The results at pH 3.0 indicate that an increasing concentration of NaCl slightly increases the helicity of (EKEK)5. One possible explanation for the observed behavior is the screening of electrostatic repulsion between like-charged Lys+ side chains. However, the helical wheel of (EKEK)5 at pH 3.0 (shown in Figure S16a) reveals a proportional distribution and spacing of similar charges on the helical surface. Hence, the charge repulsion between like charges of Lys+ should not interfere with helix formation here. This was also confirmed in our previous study on longer polypeptides, where no change in helicity was observed upon adding salt to (EKEK)25 at low pH.31 Therefore, this is unlikely to be the scenario here. A small increase in helicity after adding NaCl was observed for the host–guest E6K9 peptide (corresponding to the i, i + 3 ion pair between Glu0 and Lys+) at pH 2.5, as described in the work of Scholtz et al.28 Although the specific details of helix stabilization upon introducing NaCl into the (EKEK)5 system at low pH require further investigation, the results clearly demonstrate that ion pair formation does not have an important influence on helix formation at pH 3.0. This is because ion pair disruption due to charge screening after introducing NaCl would likely decrease helicity. The result is somewhat expected given the chosen pH, which is below the pKa value of Glu. At pH 7.0 (indicated by diamond symbols in Figure 5b), the helicity decreases to zero at cNaCl = 0.4 M. The opposing effects of ion pairs that stabilize an α-helix and those that destabilize it result in a small starting (at 0 M NaCl) helical fraction, as discussed earlier in the text. The results suggest that the addition of NaCl disrupts those ion pairs that stabilize a helix, leading to a complete loss of helicity. In the case of pH 11.5 (indicated by circle symbols in Figure 5b), the helicity remains equal to zero at all concentrations of NaCl. Therefore, unlike at pH 3.0, the (EKEK)5 polypeptides do not form helices at pH 11.5, in either the absence or the presence of NaCl. The only difference is the change in the overall charge sign of the polypeptide, with it being mostly positively charged at low pH and mostly negatively charged at high pH. The intrinsic propensity of uncharged Glu0 (pH 3.0) is higher than that of charged Glu+ (pH 7.0 and 11.5), and this effect may contribute to the presence of helical structures at pH 3.0 and their absence at pH 11.5. However, uncharged Lys0 (pH 11.5) is generally considered to have higher helical propensity than the charged version of Lys+ (expected at pH 3.0 and pH 7.0).64 Therefore, it could be expected that the higher intrinsic propensity of Lys0 at high pH would also contribute toward an increasing helical fraction. However, this has not been observed here, and the helical fraction remains zero at high pH, both in the presence and absence of NaCl. In other words, the screening of charge-repulsive electrostatic interactions has a minimal effect on helicity. The unusual helical stability of (EKEK)5 at low pH, which cannot be explained by considering only the intrinsic helical propensities of the involved amino acids, is demonstrated in Figure S17, in both the presence and absence of NaCl. The figure shows the salt-dependent experimental helical fractions and those determined from the helix prediction algorithm AGADIR. The AGADIR algorithm fails to predict higher helical fractions in both salt-free and salt-rich solutions at low pH, providing helical fractions that are significantly lower than those experimentally determined.

Figure 5.

Figure 5

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Mean residue ellipticity at 222 nm and helical fractions of (EKEK)5, (ERER)5, and (EKER)5 at three distinct pH values plotted against the concentration of NaCl. (a) MRE at 222 nm of (EKEK)5, (b) helical fractions of (EKEK)5, (c) MRE at 222 nm of (ERER)5, (d) helical fractions of (ERER)5, (e) MRE at 222 nm of (EKER)5, and (f) helical fractions of (EKER)5.

The MRE values of (ERER)5 at three different pH levels are displayed in Figure 5c, and the corresponding helicities are shown in Figure 5d. At pH 3.0, both MRE and helicity show small changes after adding NaCl. The fact that the screening of charges associated with charged Arg+ side chains at low pH does not significantly influence helicity indicates that helicity is not primarily stabilized by ion pairs formed between Arg and Glu. Similar to (EKEK)5, this is because the chosen pH is below the pKa value of Glu. The high helical content and stability of the structure with added salt are, at least partially, due to the higher intrinsic helical propensity of uncharged Glu0 (achieved at pH 3.0) compared to that of charged Glu– (achieved at pH 7.0 and pH 11.5). However, it is important to note that charged Arg+ and Lys+ do not exhibit significantly different intrinsic helical propensities.65 Yet, the helicity of Arg-rich sequences is considerably higher than that found in the Lys-rich sequences.

At the moderate and high pH values examined here (pH 7.0 and 11.5), the MRE of (ERER)5 increases, while helicity decreases with increasing NaCl concentration. The results indicate that NaCl considerably destabilizes the secondary structure of (ERER)5. NaCl salt is known for its ability to destabilize the secondary structures of various peptides. In the case of peptides where the secondary structure is stabilized by ion pairs between oppositely charged amino acids, it has been demonstrated that Na+ interacts strongly with the carboxyl groups of Glu– and Asp– as well as with the peptide backbone, leading to the destabilization of the secondary structure.44,54 The fact that the secondary structure of (ERER)5 at pH 7.0 is significantly affected by the addition of NaCl implies that the ion pairs formed between Glu– and Arg+ have an important role in the formation and stabilization of the secondary structure at this pH. Interestingly, we observe a similar trend at pH 11.5 as well. However, unlike in the case of (EKEK)5, where the Lys side chain is deprotonated, and ion pair formation between oppositely charged amino acids is not possible, the Arg side chains still retain some of their charge due to the very high pKa value of Arg. Therefore, ion pair formation can potentially stabilize the helical structure of (ERER)5 even at high pH. The results shown here suggest that by introducing NaCl, which screens electrostatic interactions, we have effectively removed an important component of the helical stabilization in the (ERER)5 system at pH 7.0 and 11.5.

The MRE and corresponding helicities of (EKER)5 are displayed in Figure 5e,f, respectively. At pH 3.0, both MRE and helicity show almost no dependence on the concentration of NaCl. At pH 7.0 and 11.5, both MRE and helicity exhibit changes with salt concentration. The (EKER)5 sequence shows a trend similar to (ERER)5 in terms of changes in MRE and the loss of helical structure upon the addition of NaCl. Although the initial (0 M NaCl) helicities at pH 7.0 and 11.5 differ between (EKER)5 and (ERER)5, the final values in Figure 5d,f (3.6 M NaCl) are nearly identical. This indicates that a similar loss of secondary structure is achieved in the two systems in the presence of a high concentration of NaCl, suggesting a high degree of side chain charge screening and ion pair disruption. The results presented here for (EKER)5 closely resemble those of (ERER)5 in the presence of NaCl, with the similarity to (EKEK)5 observed only at pH 3.0 in terms of the minimal dependence of helicity on salt concentration. They also suggest that the (i, i + 3) Glu-Arg ion pairs provide greater stabilization to the helical structures than the (i, i + 3) Glu-Lys ion pairs alone. Screening of these electrostatic interactions results in a significant loss of secondary structure. The experimental results for both (ERER)5 and (EKER)5 in the presence of NaCl were also compared with the results obtained using the helix-prediction algorithm in Figures S18 and S19, respectively. The algorithm predicts helical fractions lower than those determined experimentally at all three pH values tested. In fact, this discrepancy is more pronounced than what we observed for Lys-rich polypeptides (Figure S17).

Secondary Structure Stability of (EKKE)5 and (ERRE)5 in Salt Solutions

The salt-concentration-dependent MRE at 222 nm of (EKKE)5 at three pH values is presented in Figure 6a, and the corresponding helicities are displayed in Figure 6c. At pH 3.0, MRE decreases and helicity increases as the NaCl concentration increases. The helical wheels shown in Figure S16 suggest that charge repulsion between charged Lys+ side chains may be responsible for the inability of (EKKE)5 to form helices at a low pH in the absence of NaCl. The screening of charge repulsion between Lys+ side chains after the introduction of NaCl leads to helical formation. A similar result was reported for longer sequences in our previous publication.31 It is evident from Figure 6c that the helicity of (EKKE)5 increases with increasing NaCl concentration. However, it consistently stays lower than that of (EKEK)5 at 0 M NaCl (represented by the blue dotted line in Figure 6c). At pH 7.0, the change in MRE of (EKKE)5 with the NaCl concentration is nearly undetectable, as shown in Figure 6a. Similarly, the helical fraction is close to zero at all NaCl concentrations. The absence of salt dependence implies, as expected in this case, that salt does not disrupt any helix-stabilizing ion pairs. Additionally, it suggests that screening of charge repulsion between like-charged amino acids at pH 7.0 has no effect on helix formation. At pH 11.5, MRE is nearly independent of salt concentration, and the helicity remains close to zero at all salt concentrations. Although the charge pattern is the same as at pH 3.0, except with the opposite charge sign, the behavior differs substantially, similar to the case for (EKEK)5 at these two pH values. Additionally, the results shown in Figure 6c demonstrate that the addition of salt has almost no influence on the secondary structures in this context.

Figure 6.

Figure 6

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Mean residue ellipticity at 222 nm and helical fractions of (EKKE)5 and (ERRE)5 plotted against the concentration of NaCl. (a) MRE at 222 nm of (EKKE)5 at three distinct pH values, (b) MRE at 222 nm of (ERRE)5 at pH 3.0. (c) Helical fractions of (EKKE)5 and (ERRE)5. The blue dotted line represents the helical fraction of (EKEK)5 at pH 3.0 and 0 M NaCl. The green dotted line represents the helical fraction of (ERER)5 at pH 3.0 and 0 M NaCl.

The salt-concentration-dependent MRE of (ERRE)5 at pH 3.0 is presented in Figure 6b, with the corresponding helicities shown in Figure 6c. At low pH, (ERRE)5 does not form helices in the absence of NaCl. However, upon introducing NaCl, the helical fraction of (ERRE)5 increases and reaches the value of the (ERER)5 helical fraction in a salt-free solution at pH 3.0 (represented by the green dotted line in Figure 6c). A similar result is observed in (EKKE)5 systems at pH 3.0. Notably, the helicity of (ERRE)5 is consistently higher than that of (EKKE)5 at all tested salt concentrations. This aligns with the results shown for (EKEK)5 and (ERER)5 in Figure 5b,d, respectively, where Arg-rich polypeptides exhibited higher helicities than Lys-rich polypeptides, in both the absence and presence of NaCl. The experimentally determined helical fractions for (EKKE)5 and (ERRE)5 in salt solutions were compared with those obtained using the helix-prediction algorithm in Figures S20 and S21, respectively. The algorithm provides helical fractions close to the experimentally determined values for (EKKE)5 in the salt-free solution at pH 3.0, as well as in the NaCl solutions at pH 7.0 and pH 11.5. Additionally, it captures the increase in the helical fraction after adding NaCl at low pH for both (EKKE)5 and (ERRE)5. However, the estimated helical fractions from the algorithm at pH 3.0 are lower than those determined experimentally, similar to what we observed for (EKEK)5, (ERER)5, and (EKER)5 at low pH.

The salt-dependent study presented in the last two sections provides insights into how both charge attraction between oppositely charged side chains and charge repulsion between side chains with the same charge affect the formation of the secondary structure in the charge-patterned polypeptides studied here. It also highlights the differences between the Arg and Lys side chains. While both types of side chains represent positively charged amino acids in an ion pair, their individual contributions to helix formation are evident. The results collectively suggest that Arg side chains offer greater stabilization than Lys to the helical structures of the charge-patterned polypeptides studied here, even when ion pair formation does not influence secondary structure formation, as is the case at low pH. In the next section, we focus on the aggregation behavior of (ERRE)5 and provide a deeper understanding of the different effects of Lys and Arg on the secondary structures of charge-patterned polypeptides.

Aggregation Tendencies of (ERRE)5 Polypeptides

This section examines the previously identified aggregation behavior of (ERRE)5. The turbidity of (ERRE)5 at two distinct concentrations, dependent on pH, is shown in Figure 7a. As the pH increases, the turbidity first rises, reaching its peak at a pH close to 7.0, and then decreases without reaching the value of zero at higher pH. This similar trend is observed for (ERRE)5 at both examined concentrations (0.5 and 1.0 mM). The red dashed line represents the pKa value of Glu. The experiments conducted close to the pKa of Glu showed signs of aggregation as seen in Figure 7a. Given the predominantly positive charge of the overall polypeptide, aggregation near pKa is not anticipated, largely due to the significant charge repulsion within polypeptide species. The drop in turbidity is observed only at pH below 3.5. The shaded area in Figure 7a encompasses pH values at which the turbidity is low, indicating a lower likelihood of aggregation for the tested concentrations.

Figure 7.

Figure 7

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Turbidity measurements as a function of the pH. (a) Turbidity of (ERRE)5 and (EKKE)5 at 0.5 and 1.0 mM. The shaded area represents the range of pH values at which the aggregation of (ERRE)5 was not observed. Note that in the (EKKE)5 systems at both 0.5 and 1.0 M, the lack of aggregation results in low turbidity close to zero, with these data points overlapping. (b) Turbidity of (ERRE)5 and (ERER)5 at 0.5 mM. The red dashed line represents the pKa value of Glu.

The pH-dependent turbidity measurements of (EKKE)5 at 0.5 and 1.0 mM are illustrated in Figure 7a (represented by circles). The turbidity values show no change with pH, indicating a lack of aggregation of (EKKE)5 at the examined concentrations. This absence of aggregation persists even under charge-neutral conditions near pH 7.0. The results demonstrate an obvious difference in the aggregation tendencies between Lys-rich and Arg-rich sequences, which is particularly evident in the (EKKE)5 versus (ERRE)5 systems. The (ERRE)5 polypeptides demonstrate a clear tendency to aggregate, while the (EKKE)5 species do not exhibit aggregation. In our previous study, we reported the aggregation of (EKKE)25 systems at a high concentration and pH 7.0. However, the concentration of the systems studied in the context of aggregation in the previous work was higher than the concentration used in our current study. Furthermore, no aggregation of (EKKE)25 was observed at low or high pH.

In Figure 7b, the pH-dependent turbidity of (ERRE)5 at 0.5 mM is compared with the turbidity measurements of (ERER)5 at the same concentration. In contrast to what has been observed for (ERRE)5, the (ERER)5 polypeptides exhibit no signs of aggregation, with the measured turbidity being practically zero at all examined pH values. The results indicate that the aggregation tendency of the examined sequences is not solely dependent on the type of positively charged monomers. Instead, the charge sequence pattern significantly influences the aggregation process. Specifically, (ERRE)5 demonstrates a strong tendency to aggregate across a wide range of pH values, whereas (ERER)5 shows no aggregation across the studied pH range. The following paragraph focuses on discussing the factors influencing the aggregation tendency of (ERRE)5, as well as the absence of aggregation observed in other systems under study.

The increased occurrence of intramolecular ion pairs between Arg+ and Glu–, which stabilize helical structures as discussed in the previous sections, suggests a higher propensity for these ion pairs to form between different molecules. This propensity for intermolecular ion pairs likely promotes polypeptide aggregation. Additionally, the guanidinium group of Arg side chains possesses a unique planar geometry and exhibits low hydration both above and below the molecular plane, while retaining in-plane hydration.38,66 The low hydration observed in Arg side chains is absent in Lys side chains as NH4+ ions of Lys possess an almost spherical geometry and are fully hydrated.37,67 Hence, the stacking of Lys side chains is considered unfavorable. Studies have shown that, due to their unique geometry that decreases hydration and nonhomogeneous internal charge distribution,67 Arg side chains, unlike Lys side chains, possess the ability to be in the vicinity of other Arg monomers overcoming the evident charge repulsion.38,68 These identified factors are believed to significantly contribute to the observed tendency of Arg-rich polypeptides to aggregate under specific pH and concentration conditions, conditions under which such aggregation has not been observed in Lys-rich polypeptides. Other factors previously reported in the context of interactions between Arg-side chains, which are absent in Lys-side chains, include differences in cavitation energy (exclusion energy) and dispersion energy among like-charged ions during association.67 These were determined from molecular dynamics (MD) simulations involving both Lys and Arg side chains. Furthermore, a recent study on the reentrant behavior in liquid–liquid phase separation (LLPS) of Arg-rich protamine proteins with hyaluronic acid (HA) and poly-l-lysine with HA has underscored the significant role of hydrophobic interactions in Arg-rich systems in LLPS.37 This study highlights the differences in interactions contributing to the association of Arg- versus Lys-rich systems. Specifically, the Arg-rich protamine + HA system exhibited an increased tendency to undergo phase separation even at high salt concentrations when all electrostatic interactions were screened. In contrast, poly-l-lysine + HA showed no phase separation at high salt concentrations, as expected in a system exhibiting charge screening. The phase separation observed in the Arg-rich system at high salt concentration was attributed to the enhanced hydrophobicity of Arg side chains, a hypothesis supported by the addition of 1,6-hexanediol, known to disrupt hydrophobic interactions, which acted to dissolve the formed coacervate phase.37 Even though our study does not specifically focus on the coacervation of Arg-rich polypeptides with oppositely charged macromolecules, the highlighted study by Hong et al.37 offers additional evidence suggesting that Arg and Lys may influence macromolecular association (both self- and with other macromolecules) in distinct ways. Altogether, we believe that the above-mentioned unique features of Arg, absent in Lys side chains, including the influence of the prominent guanidinium hydrophobicity, are responsible for the aggregation of the (ERRE)5 sequence, observed even at low pH.

The question that remains is why the (ERRE)5 polypeptides form aggregates at the examined concentrations and pH while the (ERER)5 polypeptides do not. One possibility is that Arg-rich sequences containing more than one consecutive Arg within the sequence have a greater tendency to aggregate compared to sequences in which Arg monomers are interspersed with other monomers, such as Glu in our case. This implies that the clustering of Arg monomers increases the likelihood of aggregation. However, this requires further investigation, which we plan to carry out. Another explanation could be that sequences with a high charge fraction and little to no charge asymmetry might need the presence of Lys monomers to ensure solubility at charge-neutral conditions, as proposed.7 In other words, if all positively charged monomers are Arg, then the sequence might not be soluble in salt-free water at pH 7.0. Moreover, it has been shown that increasing the Lys/Arg ratio in a protein increases its solubility.69,70 However, the absence of aggregation observed in the Arg-rich and Lys-free (ERER)5 polypeptides does not fully support this alternative explanation.

Conclusions

The study elucidated the combined influence of the charge sequence pattern and the type of positively charged amino acids on the formation of secondary structures in sequence-specific polyampholytes. The effects were examined at three distinct pH values, and the stability of the secondary structure was also tested by addition of urea and NaCl. The study revealed that both the charge sequence pattern and the type of charged monomer influence secondary structure formation. (EKEK)5 exhibited pH-dependent formation of helical structure, with the highest helical fraction detected at low pH. In contrast, (EKKE)5 polypeptides failed to form helices at all examined pH values. These results align with our recently published work on longer sequences.31

Lys-to-Arg substitution demonstrated a pronounced influence on the secondary structure formation. At all examined pH values, Arg-rich polypeptides with the ERER charge pattern formed a fraction of helices that was higher than that of Lys-rich polypeptides with the EKEK charge pattern. Arg-rich polypeptides also maintained greater stability under matching urea conditions. In NaCl solutions, both Arg-rich and Lys-rich polypeptides displayed a nonmonotonic change in secondary structure with increasing NaCl concentration. The mixed sequence (EKER)5 demonstrated behavior characteristic of both Lys-rich and Arg-rich sequences, displaying the influence of both Lys and Arg on secondary structure formation. The (ERRE)5 polypeptides readily aggregated across a wide range of pH values and concentrations, as confirmed by circular dichroism (CD) analysis. CD spectra analysis revealed the β–sheet formation that can be attributed to intermolecular interactions between (ERRE)5 polypeptides (data not shown here). Aggregation was not detected only when the pH was below 3.5. In contrast, polypeptides with the same charge pattern, (EKKE)5, as well as the polypeptides with the equivalent amino acid content, (ERER)5, showed no aggregation formation under matched pH and concentration conditions. These results underscore the combined prominent effect of both the charge sequence pattern and the type of positively charged monomers on secondary structure formation.

The sequences examined here demonstrate the ability to form secondary structures, a phenomenon not commonly reported for proteins with intrinsically disordered regions. Conversely, the sequences studied here lack the ability to form the i, i + 4 ion pair, which typically stabilizes single alpha helices (SAHs). Nevertheless, our results reveal that in the case of Arg-rich polypeptides, i, i + 3 ion pairs provide significant helical stabilization that we do not observe to the same extent for Lys-rich polypeptides. This is in agreement with the MD simulations of Wolny et al.,7 which revealed a shorter lifetime and a higher frequency of formation of i, i + 3 ion pairs between Glu and Arg than i, i + 3 ion pairs between Glu and Lys. However, further investigation is needed to reveal whether this result also implies that Glu-Arg i, i + 3 ion pairs are more stabilizing than i, i + 4 Glu-Arg ion pairs, which is opposite to what has been established for Glu-Lys ion pairs.28,29

The study presented here should be relevant to future experimental, computational, and theoretical studies equally. As demonstrated in this work, a comparison of helical fractions revealed a significant discrepancy between experimentally determined helical fractions and those predicted by the helix-prediction algorithm.49 Recently, it has also been shown that special care is necessary when employing molecular dynamics (MD) simulations to accurately capture helical fractions in highly charged polypeptide sequences.71 The results presented here can inform the computational models with the aim of improving the available models. Gaining more insights into ion pair formation in specific cases of the sequences studied here through simulations would help advance our knowledge on the influence of charge sequence patterns and the type of charged monomer on the formation of secondary structures. Simulation studies could also enhance our understanding of the aggregation behavior reported for the Arg-rich (ERRE)5 sequence. Additionally, a closer examination of the interactions of urea, Na+, and Cl– with the side chains and the backbone of the examined sequences can be performed. Experimentally, exploring additional aspects could contribute further to the knowledge established here. These additional aspects include the influence of other charge sequence patterns where both i, i + 4, and i, i + 3 ion pairs would be available for helical stabilization, as well as the introduction of charge asymmetry.

Acknowledgments

J.D. and M.V.T. thank the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Materials Sciences and Engineering Division. J.D. acknowledges Dr. Wei Chen, Center for Molecular Engineering and Materials Science Division at Argonne National Laboratory for the mentorship and insightful discussions. J.D. also acknowledges Dr. Elena Solomaha at the University of Chicago Biophysics Core Facilities for the use of the CD spectrometer, Jasco Inc.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.4c00002

  • Additional figures provide further details on polypeptide characterization and experimental results; RP-HPLC chromatogram (Figures S1–S5) and ESI-MS (Figures S6–S10) of (EKEK)5, (EKKE)5, (ERER)5, (ERRE)5, and (EKER)5; sample turbidity of (ERRE)5 at varying wavelengths (Figure S11); ion pair formation between oppositely charged amino acids in sequence-specific polyampholytes (Figure S12); helicity formula vs. AGADIR predictions (Figure S13); the mean residue ellipticity at 222 nm (MRE222) ratios as a function of urea concentration (Figure S14); determination of the s-parameter (Figure S15); helical wheels of (EKEK)5 and (EKKE)5 (Figure S16); helicity of (EKEK)5, (EKKE)5, (ERER)5, (ERRE)5, and (EKER)5 in salt solutions and AGADIR helix prediction algorithm at 25 °C (Figures S17–S21) (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This work was funded by the Advanced Materials for Energy Water Systems (AMEWS) Center, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences.

The authors declare no competing financial interest.

Special Issue

Published as part of Biomacromoleculesvirtual special issue “Peptide Materials”.

Supplementary Material

bm4c00002_si_001.pdf (1.3MB, pdf)

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