pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides

Abstract

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)₃, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)₃, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)₃, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)₃; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation.

Introduction

Recently, attempts have been made to use polypeptide associations as novel bio-nanomaterials, and many new materials that use polypeptides have been reported.^1–6 Because of the regularity of polypeptide assemblies, polypeptides can be designed to form unique supermolecular formations. There are various polypeptide assemblies, including fiber,^6–8 tube,⁹ sphere,¹⁰ and vesicle.¹¹ Several of these have been characterized as valuable materials, one example being the functional hydrogel.^12–17 Several bio-nanomaterials made sensitive to various outside stimuli (e.g., pH and metal ions) by mutations in their polypeptides have been developed. Fiber-shaped associations of polypeptides have been reported in nanoscale molecules. Attempts have been made to design novel materials by using these polypeptide assemblies. For example, metal nanowires have been built by using the fiber as a scaffold and covering the fiber surface with metal.^18–21

The amphipathic α-helix is a fundamental protein structure.^22–25 With their hydrophobic surfaces, amphipathic α-helices form a bundle structure comprising two to seven molecules.^22–29 The amino acid sequences of the coiled coil–forming region consist of repeats of seven-amino-acid residues (abcdefg), in which particular positions are occupied by similar kinds of amino acid residues.³⁰ Positions a and d are occupied by hydrophobic amino acids, and the other positions are occupied by hydrophilic amino acids.^{26,28,29,31–34} Charged amino acids are often found at positions e and g. These amino acids take part in electrostatic interactions with other α-helices and stabilize the amphipathic α-helix structure.^{26,28,29,35–37} Several artificially designed polypeptides that form amphipathic α-helices have charge amino acids at positions e and g; a relationship has been reported between the electrostatic state at positions e and g and α-helix formation and thermal stability.

Over the past 20 years, artificially designed α-helix-forming polypeptides have been reported to assemble into nanoscale fibrils.^{1–5,38–61} The formation of several types of fibrous assembly, as well as changes in the shape of these assemblies, is reportedly affected by the buffer conditions. For example, Pandya et al.³⁸ designed self-assembling fiber (SAF) polypeptides with sticky ends, and Boon et al.⁴⁸ designed the polypeptide KIA13, of which shape could be varied by changing the salt concentration in the buffer. SAF polypeptides form fibers in salt-free buffer conditions; however, they cannot not form fibers in buffer with 0.5M KF³⁸ In addition, α-helix and fibrous assembly formation by KIA13 depends on the NaCl concentration.^47,48 The fibrous assemblies of KIA13 are thin under low-NaCl conditions, whereas thick fibers formed from bundles of thin fibers occur under high-NaCl conditions.⁴⁸

pH-dependent fibrous assembly formation by polypeptides has been investigated by several groups.^49,50,53 For example, Potekhin et al.⁴⁹ designed a polypeptide α-helical fibril-forming peptide (αFFP) with glutamine (Glu) at position g. It formed fibrous assemblies under acidic conditions, but it formed particle associations under neutral conditions. In contrast, αFFP polypeptide with a mutation of Glu to serine (Ser) at position g formed an α-helix and fibrous assemblies not only under acidic conditions but also under neutral or basic conditions.⁵⁰

In another example, Dublin et al.⁴ and Zimenkov et al.⁵³ designed the polypeptide TZ1H, with histidine (His) residues at the hydrophobic surface. It formed an α-helix and fibrous assemblies under neutral or basic buffer conditions, but no α-helix formation was observed under acidic buffer conditions, possibly because of a change in the electrostatic charge state of the His side chain. Therefore, α-helix and fibrous assembly formation, as well as the shapes of the fibrous assemblies, can be varied according to the electrostatic state of the polypeptide. Furthermore, Dublin et al. reported that α-helix formation of the polypeptide TZ1H depended on the Ag⁺ ion concentration.^4,54 In addition, Anzini et al. designed the peptide, TZ1C2, with Ile and Cys residues at positions a and d, respectively. The peptide has formed synthetic coiled-coil fiber without Cd (II) ion; however, with Cd (II) ion, predominant trimerization of TZ1C2 peptide has been observed.⁵⁵

We designed polypeptide α3, which was composed of three repeats of the seven-amino-acid sequence abcdefg (LETLAKA)₃.^57–60 The hydrophobic surface of α3 consisted of leucine (Leu) and alanine (Ala), and the hydrophilic surface consisted of Glu, lysine (Lys), and threonine (Thr). Positions b and f were placed on the hydrophilic surface of α3, and α3 was designed to form an intra-salt bridge between Glu at position b and Lys at position f.

α-helix formation ability of α3 depends on the polypeptide's concentration.^58–60 Transmission electron microscopic observation has shown that α3 forms long fibrous assemblies.^57,59 Almost all mutated polypeptides form fibrous assemblies under those buffer conditions in which an α-helix is formed.^58–60 The thinnest fibrous assemblies of α3 and of α3 analog polypeptides are about 20 Å wide; this width is in agreement with the width of one fiber of each bundle formed when the polypeptides form α-helix associations.^58–60

Although the 20-Å thinnest width is not affected by the buffer conditions, the association of these thinnest fibrous assemblies is affected by the pH. Flexible fibrous assemblies composed of one or two of the thinnest, mesh-like fibrous assemblies are observed at pH 2 or 12, whereas thick fibers assembled from many 20-Å-wide fibers are observed at pH 6.⁵⁹ In addition, we have found that the fibrous assemblies of α3 and different chain–length polypeptides become organized and liquid-crystallized when they are placed in a strong magnetic field for a few days.⁵⁹

Here, we investigated the relationship between the electrostatic states of an α3 polypeptide composed of three repeats of a seven-amino-acids sequence (LETLAKA) and the formation of an amphipathic α-helix or fibrous assembly, or both. We examined the properties of three kinds of polypeptide with substitutions at position b or f, or both. Polypeptide 2Sα3 had Ser substituted for Glu at position b. Polypeptide 6Sα3 had Ser substituted for Lys at position f. Polypeptide 2S6Sα3 had Ser substituted for Glu and Lys at positions b and f, respectively. The substitution positions are shown in the helical wheel of polypeptide α3 (Fig. 1), and the sequences of the substituted polypeptides are shown in Table1. We analyzed the physicochemical characteristics of position -b or -f (or both) substituted polypeptides of α3 by using their circular dichroism (CD) spectra and transmission electron microscopy.

Figure 1.

Helical wheel of the α3 polypeptide. α3 was designed to be amphipathic when it formed an α-helix. Red circles show positions b and f. Polypeptides were created with substitutions of Glu at position b or Lys at position f (or both) with Ser.

Table 1.

Amino Acid Sequences of α3 and Position b- or f- (or both) Substituted Polypeptides

α3	LETLAKA LETLAKA LETLAKA
2Sα3	LSTLAKA LSTLAKA LSTLAKA
6Sα3	LETLASA LETLASA LETLASA
2S6Sα3	LSTLASA LSTLASA LSTLASA

We examined the properties of three polypeptides with substitutions at position b or f, or both. Polypeptide 2Sα3 had Ser substituted for Glu at position b. Polypeptide 6Sα3 had Ser substituted for Lys at position f. Polypeptide 2S6Sα3 had Ser substituted for Glu and Lys at positions b and f, respectively.

Results and Discussion

Preparation of position b- or f- (or both) substituted polypeptides

We prepared polypeptides with Ser substitutions at position b or f, or both, by using the gene engineering methods we had used to prepare polypeptide α3 in our laboratory (see Materials and Methods). However, the growth rates of the transformed E. coli expressing position b- or f- substituted polypeptides were slow in comparison with that of E. coli expressing fusion protein of adenylate kinase (ADK) with α3; furthermore, the quantity of expressed fusion protein of ADK and position b- or f- substituted polypeptide was very small. The quantity of expressed fusion protein of 2S6Sα3, in particular, was extremely low. Therefore, we prepared this polypeptide by chemical synthesis instead (see Materials and Methods).

CD spectra measurement

To examine the secondary structures of position b- or f- (or both) substituted polypeptides, we measured the CD spectra of the substituted polypeptides in 0.1M KCl 10 mM citrate buffer (pH 2), 0.1M KCl 10 mM phosphate buffer (pH 6), or 0.1M KCl 10 mM borate buffer (pH 12) at a polypeptide concentration of 150 µM at 30°C (Fig. 2).

Figure 2.

CD spectra of α3 and of position b- or f- (or both) substituted polypeptides. CD spectra of the polypeptides were measured in a solution containing 0.1M KCl and 150 μM of each polypeptide at 30°C.

α3 formed an α-helix under all pH conditions in 0.1M KCl [Fig. 2(A)]; the α-helix was the most stable at pH 6. 2Sα3 and 6Sα3 formed α-helices under those buffer conditions in which electrostatic repulsion was suppressed [Fig. 2(B,C)]; these were 0.1M KCl 10 mM borate buffer (pH 12) in the case of 2Sα3, and 0.1M KCl 10 mM citrate buffer (pH 2) in the case of 6Sα3. 2S6Sα3 formed an α-helix in 0.1M KCl 10 mM borate buffer (pH 12) [Fig. 2(D)]. At pH 6, 2S6Sα3 formed a pattern in between randomness and an α-helix, and at pH 2 no helix formed. The α-helix-formation ability of 2S6Sα3 was thus higher under basic conditions than under acidic conditions.

We then measured the CD spectra of the substituted polypeptides, with changes in pH (2, 6, 12), KCl concentration (0.1M or 1M), and polypeptide concentration (about 25–250 μM), to determine the optimum conditions for helix formation. We plotted the absolute values of mean residue molar ellipticity [θ] at a wavelength of 222 nm against the concentration of each sample under specific conditions (Supporting Information Fig. S1). The absolute values of mean residue molar ellipticity [θ] at 222 nm for position b- or f- (or both) substituted polypeptides depended on the polypeptide concentration at which they formed α-helix buffer. Under 0.1M KCl, α-helix formation by 2Sα3 was observed at the concentration of higher than about 25 μM in 10 mM borate buffer (pH 12), and α-helix formation by 6Sα3 was observed at concentration of 6Sα3 higher than 25 μM in 10 mM citrate buffer (pH 2). Under 1M KCl, α-helix formation by 2Sα3 and 6Sα3 depended on their concentrations at all pH values. The absolute value of the mean residue molar ellipticity [θ] of 2S6Sα3 at 222 nm depended on the polypeptide concentration, except under 1M KCl 10 mM borate buffer (pH 12) conditions; the α-helix-formation ability of 2S6Sα3 at pH 12 was higher than that at the other pHs. For example, the absolute value of the mean residue molar ellipticity [θ] at 222 nm of 150 μM 2S6Sα3 containing 0.1M KCl was about 3,400, 6,300, and 15,000, at pH 2, 6, and 12, respectively. The concentration of 2S6Sα3 at which the α-helix structure could be stabilized was lower in 1M KCl than in 0.1M KCl.

Observation of fibrous assembly by electron microscopy

To examine fibrous assembly formation, we observed the assembly of position b- or f- (or both) substituted polypeptides under a JEM 1010 transmission electron microscope (JEOL) using 2.5% uranyl acetate (Fig. 3). Electron microscopic observations were made at the same (150 μM) polypeptide concentration at various pH conditions (2, 6, and 12) and with 1M KCl.

Figure 3.

Electron photomicrographs of position b or f (or both) substituted polypeptides. The buffer conditions for electron microscopic observation were 1M KCl 10 mM citrate buffer (pH 2), 1M KCl 10 mM citrate buffer (pH 6), or 1M KCl 10 mM borate buffer (pH 12). The polypeptide concentration was 150 μM. Fibrous assemblies of polypeptides were stained with 2.5% uranyl acetate solution. Scale bar represents 50 nm.

Fibrous assemblies were observed under the same buffer conditions at which α-helices were formed by the three polypeptides, suggesting that not only α-helix formation but also fibrous assembly formation depended on pH. The fibrous assemblies of 2Sα3 formed tangled mesh-like nets at pH 2 [Fig. 3(A)]; the thickness of the fibrous assembly at pH 2 was about 40 Å (42.3 ± 1.8 Å). At pH 6 and pH 12, the fibrous assemblies of 2Sα3 tended to form thick bundles consisting of many thin fibers [Fig. 3(B,C)]. The fibrous assemblies of 6Sα3 formed mesh-like nets at both pH 2 and pH 6 [Fig. 3(D,E)]—similar to the fibrous assemblies of 2Sα3 under acidic conditions [Fig. 3(A)]. The thickness of the fibrous assemblies at pH 2 was about 40 Å (43.3 ± 1.6 Å). Short fibers of the thickness 40 Å were formed at pH 12 [Fig. 3(F)]. In the case of 2S6Sα3, were observed at all pHs [Fig. 3(G–I)]. The thicknesses of fibrous assemblies of 2S6Sα3 at pH 2 were 40 Å (38.1 ± 1.9 Å). However, the frequencies of occurrence of fibrous assemblies were lower at pH 6 and 12 than at pH 2, and the fibrous assemblies were shorter than those of the other polypeptides [Fig. 3(G,I)].

The width of the thinnest fibers of the three substituted polypeptides was calculated to be about 20 Å, and this width did not change with changes in the buffer conditions (i.e., the pH or KCl concentration). For example, the thicknesses of the thinnest fibrous assemblies of 2Sα3, 6Sα3, and 2S6Sα3 in 10 mM citrate buffer (pH 6) containing 0.1M KCl were 20.3 ±1.4 Å, 19.5 ± 1.4 Å, and 19.9 ± 1.5 Å, respectively. In addition, the association states of the thinnest fibrous assemblies of position b- or f- (or both) substituted polypeptides were distinct from that of α3: in 2Sα3, thick fibrous assemblies bundles, of which thicknesses of bundles were about 20–30 nm, were formed by association under neutral and basic conditions, whereas in 6Sα3 and 2S6Sα3, fibrous assemblies predominantly 40 Å thick formed under all buffer conditions.

In the case of 6Sα3 and 2S6Sα3, formation of thick and long fibers from the association of many 20-Å fibers was not observed. Taking these results together, we revealed that the outside of the α-helix (consisting of the hydrophilic surface) was involved in the association of α-helix fibers.

Also, the results suggested that the association states of the thinnest fibrous assemblies were related to the charge states of the amino acids, especially in the case of the Lys residue at position f. Several groups have reported multimerization of fibers by interactions between individual fibers^40,41,51,56 Kajava' s group⁵¹ and Dong's group⁵⁶ found that fibrous assembly formation was due to the interaction of hydrophilic surfaces. In addition, Papapostolou's group⁴⁰ succeeded in controlling fiber thicknesses through amino acid substitutions in the polypeptide SAFs they designed. Furthermore, Gribbon et al.,⁴¹ in their designed MagicWand series of polypeptides, found that cation–π interactions between the amino acid residues located at positions b and f were important for fibrous assembly formation and for the thickness of fibrous assemblies.

Effect of pH on α-helix-formation ability of mutated polypeptides

To examine the effect of only pH value on α-helix formation of position b and/or position f substituted polypeptides, we measured the CD spectra of α3 and the three kinds of substituted polypeptide under conditions of 0M KCl and 150 μM of each polypeptide, at 30°C, and with a variable pH (2–12) [Fig. 4(A)]. α3 formed an α-helix at pH 4–11; its α-helix-formation abilities were low under extreme acidic conditions and basic conditions, where the charges of Glu and Lys were repulsed in the polypeptide, but not under neutral conditions.

Figure 4.

Effect of pH value on α-helix and fibrous assembly formation ability of α3 and of position b- or f- (or both) substituted polypeptides. (A) Mean residue molar ellipticity [θ] value at 222 nm in the CD spectra of position b- or f- (or both) substituted polypeptides with changes in pH value (from 2 to 12). CD spectra of polypeptides were measured at 30°C in a solution containing 150 μM of each polypeptide. (B) Fibrous assembly formation. The buffer conditions for electron microscopic observation were 0M KCl 10 mM citrate buffer (pH 2) or 0M KCl 10 mM borate buffer (pH 12). The polypeptide concentration was 150 μM. Fibrous assemblies of polypeptides were stained with 2.5% uranyl acetate solution. Scale bar represents 50 nm.

The α-helix-formation ability of 2Sα3 increased gradually above pH 8, and the absolute mean residue ellipticity [θ] at 222 nm did not change above about pH 10. 2S6Sα3 showed similar pH dependency. In 2Sα3 and 2S6Sα3, the midpoint of α-helix formation against pH was approximately 9—smaller than the pKa of the Lys side chain. These results suggest that the electrostatic state at the amino terminus and the local structure around the Lys residues contribute to α-helix formation by 2Sα3 and 2S6Sα3. We considered that the stabilization of the α-helix of 2S6Sα3 under basic conditions might be due to suppression of the electrostatic repulsion of the Leu residue located at the amino terminal, because there is no Lys residue in 2S6Sα3 and Leu residues play important roles in amphipathic α-helix formation. For example, Fairman et al. have described the relationship between the electrostatic charge states at the termini and α-helix formation.⁶²

We found here that the charge state of the Leu residue (which is important for amphipathic α-helix formation) located at the terminus affected amphipathic α-helix formation.

In contrast to the other two polypeptides, 6Sα3 formed an α-helix at pH values lower than 5.5, and the absolute value of the mean residue ellipticity [θ] at 222 nm increased gradually at pH values lower than 4 [Fig. 4(A)].

Several papers have investigated the correlation between stabilization of the α-helix with protonation of the side chain of Glu.^31,35–37 We used electron microscopy to examine α3, 2Sα3, and 6Sα3 fibrous assembly formation under the conditions of polypeptide concentration 150 μM and 10 mM citrate buffer (pH 2) or 10 mM borate buffer (pH 12), without KCl. 2Sα3 formed fibrous assemblies only at pH 12 without KCl, whereas 6Sα3 formed fibrous assemblies only at pH 2 without KCl [Fig. 4(B)]. The α-helix-formation abilities of α3 are lower at pH 2 or 12 than at pH 6 [Fig. 4(A)]; nevertheless, α3 forms fibrous assemblies both at pH 2 and pH 12 without KCl (data not shown).

The αFFP designed by Potekhin et al.⁴⁹ formed an α-helix and fibrous assemblies only under acidic conditions because of the electrostatic state of Glu at position g. They prepared mutants of αFFP in which Glu at position g was replaced by Gln or Ser; the substituted polypeptides formed α-helices and fibrous assemblies regardless of the pH range (pH 2.5–11).⁵⁰ We revealed here that fibrous assembly formation could be controlled by changing the electrostatic state of α3 at position b or f under various pH conditions.

Effect of KCl concentration on α-helix-formation ability of 2Sα3 and 6Sα3

Next, we measured the CD spectra of 2Sα3 and 6Sα3 in 10 mM citrate buffer (pH 2) or 10 mM borate buffer (pH 12), with a polypeptide concentration of 150 μM, a temperature of 30°C, and a variable KCl concentration (0–1M), to determine the optimum KCl concentration for α-helix formation. We plotted the mean residue molar ellipticity [θ] at 222 nm against the concentration of KCl under specific conditions (Supporting Information Fig. S2).

For 2Sα3 in 10 mM citrate buffer (pH 2), the absolute value of the mean residue ellipticity [θ] at 222 nm did not change below 100 mM KCl, whereas above 100 mM KCl the ellipticity increased gradually. In contrast, 6Sα3 formed an α-helix even at 0 mM KCl, and as the concentration of KCl increased from 0 to 100 mM the absolute value of the mean residue ellipticity [θ] at 222 nm increased. Above 100 mM the ellipticity slightly decreased. For 2Sα3 in 10 mM borate buffer (pH 12), an α-helix was formed with 0 mM KCl; then, as the KCl concentration increased, the absolute value of the ellipticity [θ] at 222 nm decreased wavered up and down but showed an overall slow decrease. In contrast, the absolute value of the mean residue ellipticity of 6Sα3 at 222 nm increased gradually from above 200 mM KCl.

From these results, we concluded that the lowest KCl concentration for α-helix formation under electrostatic repulsion pH conditions was approximately 100 mM for 2Sα3 and approximately 200 mM for 6Sα3.

This difference in the lowest KCl concentration at which the polypeptide can form an α-helix because of the presence of the Lys side chain might be due to the existence in 2Sα3 of two methylene groups longer than the Glu side chain between the charged group and the α carbon; thus α-helix formation in 2Sα3, unlike that in 6Sα3, is not affected by electrostatic repulsion of the amino acid side chain.^63,64 In addition, 6Sα3 has Glu as a charged amino acid at position b and 2Sα3 has Lys as a charged amino acid at position f. It is plausible that the difference in α-helix formation abilities originates from the fact that position f is more exposed than position b.²⁹

Observation of the fibrous assemblies of the respective polypeptides revealed that, for 2Sα3, fibrous assemblies were observed in 10 mM citrate buffer (pH 2) containing 100 mM KCl, under which conditions 2Sα3 forms an α-helix (Fig. 2 and Supporting Information Fig. S1). In 10 mM borate buffer (pH 12), 2Sα3 formed an α-helix, and its fibrous assemblies were observed without KCl (Fig. 4). 2Sα3 was able to form fibrous assemblies at any KCl concentration, and the morphology of the fibrous assemblies did not change with changes in the KCl concentration (data not shown).

The fibrous assemblies of 2Sα3 were observed to be thick bundles at pH 6 and 12, corresponding to bundles of several dozen of the thinnest fibers (20 Å). The lengths of the fibrous assemblies ranged from chiefly about 200 to 500 nm and the length of long fibrous assemblies was 1 μm. And thicknesses of bundles were 20–30 nm at pH 6 and 12. For 6Sα3 in 10 mM citrate buffer (pH 2), an abundance of mesh-like fibers was observed at KCl concentrations ranging from 0 to 1M [Figs. 3(D) and 4(B)].

At low KCl concentrations, virtually none of the fibers tended to bundle; however, an increase in the concentration of KCl caused the fibers to assemble into thick bundles (data not shown). At KCl concentrations greater than 200 mM, 6Sα3 formed a mesh of fibers formed in 10 mM borate buffer (pH 12). From these results, we concluded that α-helix and fibrous assembly formation by 2Sα3 and 6Sα3 under buffer conditions that suppressed electrostatic repulsion did not depend on the KCl concentration; under buffer conditions where there was a repulsive electrostatic charge they did depend on the KCl concentration.

Frost et al.⁴⁸ similarly found that KIA13, the polypeptide they designed, which was composed of Lys, isoleucine, and Ala residues, formed an α-helix and fibrous assemblies in an NaCl concentration-dependent manner.

pH-dependent reversibility of α-helix and fibrous assembly formation by 2Sα3 and 6Sα3

To examine whether or not the formation of the α-helix and fibrous assemblies of 2Sα3 and 6Sα3 was reversible, we measured CD spectra with alternate addition of HCl and KOH solutions into the polypeptide solution every five minutes (Fig. 5). We did not examine the reversibility of fibrous assembly formation in the case of α3 and 2S6Sα3, because the pH specificity of their fibrous assembly formation was low.

Figure 5.

Reversibility of formation of α-helix and fibrous assemblies of 2Sα3 and 6Sα3 with changes in pH. (A) CD spectral patterns of 2Sα3 and 6Sα3 in solutions to which HCl and KOH were added for the first time. CD spectra were measured at 30°C in a solution containing 150 μM of each polypeptide. (B) Mean residue molar ellipticity [θ] values at 222 nm in CD spectra of 2Sα3 and 6Sα3 in solutions to which HCl and KOH were added alternately. CD spectra of polypeptides were measured at 30°C in a solution containing 150 μM of each polypeptide. Odd numbers on x-axis: addition of 2 N HCl to polypeptide solution; even numbers on x-axis: addition of 2 N KOH to polypeptide solution. (C) Electron micrographs of 2Sα3 and 6Sα3 to which HCl or KOH was added. “HCl 1” and “KOH 1” indicate that HCl or KOH was added once, and HCl 2 and KOH 2 indicate that they were added twice. The polypeptide concentration was 150 μM. Fibrous assemblies of polypeptides were stained with 2.5% uranyl acetate solution. Scale bar represents 100 nm.

First, we added 1 μL of 2M HCl to 250 μL of a solution comprising 150 μM polypeptide and then immediately measured the CD spectra. Next, we added 2 μL of 2M KOH to the polypeptide solution. After the addition of a further 2 μL of 2M HCl and 2 μL of 2M KOH to the polypeptide solution alternately, we again immediately measured the CD spectra of the solution [Fig. 5(A)]. After the addition of HCl (acidic conditions) to the polypeptide solution, 2Sα3 was not able to form a secondary structure, whereas 6Sα3 formed an α-helix. Next, after the addition of KOH (basic conditions), 2Sα3 formed an α-helix but 6Sα3 had no secondary structure. The secondary structure of 2Sα3 was disordered under acidic conditions, but an α-helix was formed under basic conditions. In contrast, 6Sα3 formed an α-helix under acidic conditions, but the structure of 6Sα3 was disordered under basic conditions. Thus α-helix formation by 2Sα3 and 6Sα3 was reversible against pH [Fig. 5(B)]. The secondary structures of these polypeptides mostly changed within several minutes after each addition of HCl or KOH.

The absolute value of the mean residue molar ellipticity [θ] at 222 nm increased with increasing number of times the HCl and KOH were added, not under only α-helix-forming conditions but also even under disordered conditions. This might have been due to the increase in the concentration of KCl generated by the addition of HCl and KOH and the decrease in the apparent concentration of the non-fibrous peptide. The change in mean residue molar ellipticity [θ] at 222 nm in the case of 2Sα3 was larger than that for 6Sα3.

In addition, electron microscopic analyses showed that, for 2Sα3, bundles composed of fibers of approximately 20 Å were present under basic conditions but disappeared under acidic conditions [Fig. 5(C)]; however, mesh-like fibrous assemblies were present when the number of additions of HCl and KOH was four or more (data not shown). In contrast, in the case of 6Sα3, fibers were observed piled up into a mesh under acidic conditions but disappeared under alkaline conditions [Fig. 5(C)]; however, after more than two additions of KOH, very short fibrils were observed fairly infrequently (data not shown). These might have originated from KCl generated by the addition of HCl and KOH. From these results, we concluded that 2Sα3 and 6Sα3 showed fibrous assembly formation reversible by pH changes, despite limited addition of acid and base. Formation and disappearance of the 2Sα3 and 6Sα3 fibers occurred within several minutes.

In many studies, in vitro amyloid fiber formation revealed by thioflavine T binding assays has demonstrated that amyloid fiber formation is generally dependent on the concentration of peptide, protein, salt concentration, or pH.^65–68 These assays have also shown that fiber formation has a lag time, and that amyloid fiber formation takes from several tens of minutes to several hours.^65–68

In addition, Bromley's group described peptide SAFs (SAF-p1 and SAF-p2a) at the concentration of each peptide was 100 μM that needed 10–30 min for thick, long fiber formation after each peptide was mixed.⁴³

In conclusion, we obtained the following results: 2Sα3 and 6Sα3, which had different numbers of charged amino acids compared with those of α3, formed α-helices and fibrous assemblies under pH conditions that suppressed electrostatic repulsion (2Sα3: pH 12; 6Sα3: pH 2) in 0.1M KCl. α-helices were formed at any of the tested pH values in 1M KCl.

We successfully showed that the characteristics of α3, which forms an α-helix and fibrous assemblies over a wide pH range, were changed such that pH-dependent and reversible α-helix and fibrous assembly formation occurred upon the substitution of charged amino acids with Ser residues at position b or f. 2S6Sα3 formed an α-helix under basic conditions; this might have been due to the state of electrostatic charge of the Leu residue located at the amino terminus.

Finally, we showed that fibrous assembly formation by α3 fibers could be controlled by pH change (i.e., acidic or alkaline conditions) through external stimuli. We expect that mutated polypeptides will be applicable to the design of novel nanomaterials such as pH-dependent biosensors in the near future. It should be noted that the present finding is consistent with the principle of α-helical formation in proteins. Finally, evaluation of structural characteristics of the targeted proteins in the fibers would provide further insights into their unique properties designed in this report.

Materials and Methods

Preparation of polypeptides by gene engineering

Position b- or f- (or both) substituted polypeptides, with the exception of 2S6Sα3, were prepared by means of a gene engineering method. The polypeptides were expressed as fused proteins with the N-terminal part of porcine ADK, because the expression efficiency of the vector constructed for this protein (i.e., ADK) is high in Escherichia coli.⁶⁹ The DNA strand corresponding to the inner part of ADK was added to the sequence ATG coding methionine and thereafter to the DNA sequence corresponding to the designed polypeptide in order to cut the polypeptide out of the fused protein product by means of cyanogen bromide (BrCN) treatment. The DNA chains coding the polypeptides consisted of the most suitable codons in E. coli; complementary DNA chains were designed. These DNAs were synthesized by Nissinbo. Double-stranded DNAs were prepared by annealing.

Each chemically synthesized double-stranded DNA was inserted between restriction sites EcoRI and BamHI of the cloning vector pUC18. After the base sequence had been checked, the DNA was treated with three enzymes, namely EcoRI, PstI, and the Klenow fragment of DNA polymerase I. The DNA fragments thus obtained were purified and inserted between restriction sites SmaI and PstI of the expression vector pMKAK3 to prepare the expression vector pMK peptide that we used here. The expression vector pMKAK3 is a circular DNA that is able to express large amounts of ADK as inclusion bodies in E. coli.

Escherichia coli strain JM109 transformed with pMK peptide was incubated at 37°C for 18 h in Luria Bertani medium containing 50 mg/L of ampicillin. Cells were collected by centrifugation at 2000g for 10 min at 4°C. The harvested cells were washed twice with TE (10 mM Tris-HCl; pH 8) buffer containing 1 mM EDTA. Washed cells were suspended in TE buffer containing 1 mM EDTA and then disrupted by sonication on ice. The fused protein was collected as inclusion bodies by centrifugation at 8000g for 10 min at 4°C. To split off the N-terminal part of ADK from the designed polypeptide, the inclusion bodies were incubated in 70% formic acid, 2% BrCN, and 1% 2-mercaptoethanol at 37°C for 48 h. They were then dialyzed against MilliQ water at 4°C for 8 h and then against 10 mM Tris-HCl buffer (pH 8.5) at 4°C overnight.

The precipitate was centrifuged off at 8000g for 10 min at 4°C. The dialyzed solution was applied to a diethylaminoethyl column (ø 3 × 10 cm) (Whatman, UK) and then washed with 200 mL of 0.01M Tris–HCl buffer (pH 8.5) at a flow rate of 1 mL/min. The designed polypeptides were eluted with 200 mL of 0.5M NaCl in 0.01M Tris–HCl buffer (pH 8.5). The polypeptide-containing fractions were further purified by reverse-phase high-performance liquid chromatography (HPLC) on a C4 column (C4 P-300-5, ø 4.6 × 150 mm; Tokyo Kasei, Japan). A linear gradient of solvents A and B was applied for 60 min. Solvent A comprised 0.1% trifluoroacetic acid (TFA) (v/v) in MilliQ water, and solvent B comprised 0.1% TFA (v/v) and 80% acetonitrile (v/v) in MilliQ water. The polypeptide-containing fractions were collected and lyophilized. Each polypeptide was dissolved in MilliQ water and then purified by reverse-phase HPLC on a C18 column (L-column ODS, C18, ø 4.6 × 150 mm; Chemicals Inspection & Testing Institute, Japan). The solvents and the gradient conditions were the same as those used in the case of the C4 column. The purified polypeptide was lyophilized and then dissolved in MilliQ water. It was stored in a deep freeze at −30°C.

Preparation of polypeptides by chemical synthesis

Because polypeptide 2S6Sα3 was not able to be prepared by using a gene engineering method (see above, we synthesized it by using a standard 9-fluorenyl-methoxycarbonyl (Fmoc) solid-phase method in a Kokkusan peptide synthesizer (Kokusankagaku, Japan). Fmoc amino acids and all other reagents were purchased from Kokusankagaku. Fmoc amino acid coupling reactions were performed for 1.5 h at room temperature in N,N-dimethylformamide (DMF) containing N-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide. The Fmoc-group removal reaction was performed in a 20% piperidine–DMF solution for 3 min at room temperature. After all the reactions had been performed, the resin was washed with methanol and then dried with an aspirator. Polypeptides were split from the resin by incubation for 2 h at room temperature with the reaction solution, which consisted of TFA containing 15% thioanisole (v/v) and 2.5% m-cresol (v/v). TFA was removed from the reaction solution with an evaporator, and then the reaction solution was mixed with cold diethylether (about 300 mL). The crude polypeptides were collected as a white precipitate on a glass filter and then purified by size-exclusion chromatography on G-10 gel (Pharmacia, Sweden). The polypeptide-containing fractions were purified further by reverse-phase HPLC. The method of purification by reverse-phase HPLC was the same as that in the case described in the preceding section on preparation of polypeptides by means of gene engineering. The fractions containing the polypeptides were lyophilized, and the purified polypeptides were stored at −30°C until use.

Determination of polypeptide concentrations

The concentration of each polypeptide was determined by amino acid composition analysis with an Amino Acid Analyzer L-8500 (Hitachi, Japan) after hydrolysis in 20% (v/v) HCl at 110°C for 24 h under vacuum.

CD spectra measurements

CD spectra of the polypeptides were measured with a Circular Dichroism Spectrophotometer J-720 (Jasco, Japan). The light-path length of the quartz cell was 1 mm. The temperature was regulated by circulation of electrostatically controlled water through a jacket surrounding the cell.

Electron microscopic observation

Fibrous association of the polypeptides was observed by transmission electron microscopy. An aliquot of a solution comprising 150 μM polypeptide sample in 10 mM citrate buffer (pH 2 or 6) or 10 mM borate buffer (pH 12) containing various concentrations of KCl was mounted on a grid with a film of carbon-coated Parlodion, and then the grid was washed with the same buffer as used for the sample. Excess solution was removed with filter paper. The grid was then negatively stained with 2.5% uranyl acetate.

The sample-mounted grid was examined under a transmission electron microscope (JEM 1010; JEOL, Japan) at an accelerating voltage of 80 kV. Electron micrographs were recorded on Fuji Electron Microscopic Film FG (11.8 × 8.2 cm) (FujiFilm, Tokyo, Japan).

Glossary

ADK

adenylate kinase

Ala

alanine

BrCN

cyanogen bromide

CD

circular dichroism

DMF

N,N-dimethylformamide

Fmoc

9-fluorenyl-methoxycarbonyl

Gln

glutamine

Glu

glutamic acid

Gly

glycine

His

histidine

HOBt

N-hydroxybenzotriazole

HPLC

high-performance liquid chromatography

Leu

leucine

Lys

lysine

SAF

self-assembling fiber

Ser

serine

TFA

trifluoroacetic acid

Thr

threonine.

Supporting Information

Additional Supporting Information may be found in the online version of this article.