Rapid Communication

J. Biochem. 144, 15–19 (2008)
doi:10.1093/jb/mvn051

Requirement of Ala Residues at g Position in Heptad Sequence ofa-Helix-forming Peptide for Formation of Fibrous Structure
Gaku Aoki1, Toyo K. Yamada1, Mayu Arii2, Shuichi Kojima2,* and Tadashi Mizoguchi1
1

Department of Physics; and 2Institute for Biomolecular Science, Faculty of Science, Gakushuin University,
Mejiro, Tokyo 171-8588, Japan
Received March 4, 2008; accepted April 8, 2008; published online April 16, 2008

One feature of the a3-peptide, which has the amino acid sequence of (Leu-Glu-ThrLeu-Ala-Lys-Ala)3, that distinguishes it from many other a-helix-forming peptides is
its ability to form fibrous assemblies that can be observed by transmission electron
microscopy. In this study, the effects of Ala!Gln substitution at the e (5th) or g (7th)position in the above heptad sequence of the a3-peptide on the formation of a-helix
and fibrous assemblies were investigated by circular dichroism spectral measurement and atomic force microscopy. The 5Qa3-peptide obtained by Ala!Gln substitution at the e position of the a3-peptide was found to form very short fibrils withlong-elliptical shape, whereas the 7Qa3-peptide with Gln residues at the g position
lost its ability to form such assemblies, in spite of a-helix formation in both peptides;
the stabilities of both peptides decreased. These results indicate that Ala residues at
the g position in the heptad sequence of the a3-peptide are key residues for theformation of fibrous assemblies, which may be due to hydrophobic interactions
between a-helical bundle surfaces.
Key words: a-helix, atomic force microscopy, circular dichroism spectra, fibre
formation, heptad sequence.
Abbreviations: AFM, atomic force microscopy; TEM, transmission electron microscopy.

dipole of the a-helix, the stabilization of the a3-peptide bysequence reversal may be specific to only fibre-forming
peptides. On the other hand, the a-helix and fibrousassemblies of the a3-peptide are destabilized by substitutions of Leu residues on the hydrophobic surface withless hydrophobic amino acids, possibly owing to thedecrease in the degree of hydrophobic interactions (33).
This is a general feature of peptides that form multimeric a-helical bundle structures.
In this study, we focused on Ala residues at the e (5th)and g (7th) positions in the heptad sequence of thea3-peptide. Since peptides with charged residues (Gluand Lys) at these two positions form a two-stranded
coiled-coil structure (11, 14), it is suggested that Ala
residues at both or either position in the a3-peptide are
required for the formation of fibrous structures. Thus, we
substituted these Ala residues of the a3-peptide with lesshydrophobic Gln residues, since Gln is a polar amino acidwith the strongest a-helix-forming tendency among noncharged and non-hydrophobic amino acids.
a3-peptide variants with Gln residues at the e or g
position in the heptad sequence of the a3-peptide, namely
the 5Qa3- and 7Qa3-peptides (Fig. 1), respectively, werechemically synthesized and purified using reverse-phase
HPLC with an acetonitrile gradient in 0.1% trifluoroacetic acid. Their concentrations were determined by aminoacid composition analysis after hydrolysis with 5.7 N HCl
at 1108C for 24 h in vacuo.
The CD spectra of the 5Qa3- and 7Qa3-peptides were
measured using a JASCO J-720 spectropolarimetre ina neutral pH buffer at 308C to investigate the effectsof Ala!Gln substitutions at the e or g position in the

The a-helix is a secondary structure of proteins that
contributes to the stability and folding of proteins. Its
sequence-stability relationship, as well as several interactions between side chains and the intrinsic helixforming tendency of amino acids, has been extensively
studied (1–19). We previously designed and synthesized
an amphipathic 21-residue peptide (a3-peptide) withthree repeats of the seven-residue (heptad) sequence
Leu-Glu-Thr-Leu-Ala-Lys-Ala, anticipating that it will
form an a-helical bundle structure through hydrophobic
interactions between Leu residues. We found that the a3peptide exhibits a concentration-dependent stabilizationof its a-helix, suggesting the formation of oligomers (20).
Unexpectedly, we demonstrated that the a3-peptide form
fibrous assemblies that can be observed by transmission
electron microscopy (TEM) (21). To our knowledge, thismight have been the first report on the formation offibrous assemblies by a de novo-designed a-helical short
peptide, in contrast to many reports on the formation ofamyloid mainly composed of b-sheets (22–31). Thus, wehave synthesized several variants of the a3-peptide toinvestigate the relationship between the sequence,
a-helix stability and formation of fibrous assemblies.
When the sequence of the a3-peptide was reversed, theresultant r3-peptide formed a very stable a-helix andlong fibres (32). Since the a-helix, which does not form
fibrous assemblies, is generally destabilized by sequence
reversal through electrostatic repulsion with an intrinsic*To whom correspondence should be addressed. Tel: +81-3-39860221, ext. 6505, Fax: +81-3-5992-1034,
E-mail: shuichi.kojima@gakushuin.ac.jp
Vol. 144, No. 1, 200815

ß 2008 The Japanese Biochemical Society.

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G. Aoki et al.

Fig. 1. Helical wheel representations of (A) a3-, (B) 5Qa3and (C) 7Qa3-peptides. The 5Qa3- or 7Qa3-peptide wasproduced by substituting Ala residues at the e (5th) or g (7th)position in the heptad sequence of the a3-peptide.

Fig. 2. (A) CD spectra of 5Qa3-peptide (black line) and
7Qa3-peptide (gray line) at 200 kM and (B) peptide
concentration dependence of [h] at 222 nm of thea3-peptide (open square), 5Qa3-peptide (open triangle)
and 7Qa3-peptide (filled triangle) in 10 mM phosphatebuffer (pH 6.0) containing 0.1 M KCl. After the concentrationsof the peptides were determined by amino acid composition
analyses, the CD spectra at various peptide concentrations were
measured at 308C using a JASCO J-720 circular dichroism
spectropolarimetre with a path length of 1 mm; the results are
expressed as mean residue molar ellipticity.

heptad sequence of the a3-peptide on the a-helix stabilityof the a3-peptide. It was found that both peptides showed
CD spectra that indicate a-helix formation (Fig. 2A), as
in the case of other variants of the a3-peptide. However,since the helix contents of both peptides were lower thanthat of the a3-peptide, the concentration dependences of[] at 222 nm for the two peptides were investigated andcompared with that of the a3-peptide.
As shown in Fig. 2B, the [] values of the 5Qa3- and7Qa3-peptides increased as peptide concentration
increased, which strongly indicates that the a-helices ofthese peptides are stabilized by oligomerization, as in thecase of other variants of the a3-peptide. However, thea-helix of the a3-peptide was destabilized by the substitution of Ala residues at the e position in the heptad
sequence with Gln residues, and much more prominently
by the substitution at the g position. As a result, among
the peptides examined, the 7Qa3-peptide had the most
unstable a-helix. The helix contents of the a3-, 5Qa3- and7Qa3-peptides at about 200 mM, which were estimatedusing the equation []222 nm/ {40,000(12.5/n) + 100T},
where n = 21 and T = 30 (34), were 59, 39 and 27%,
respectively.
Since the a3-peptide was demonstrated to form fibrils
5–10 nm in width and intermediate in length that can beobserved by TEM in a neutral pH buffer, we observed
such fibrous structures of the a3-peptide in air at 208C
by atomic force microscopy (AFM, JEOL-JSTM-4200D),
as well as those of the 5Qa3- and 7Qa3-pepitdes todetermine the effects of Ala!Gln substitution in thea3-peptide on the formation of fibrous assemblies. AFM
in the tapping mode was performed with Si cantilevers(spring constant: 1.38 N/m, resonance frequency: 74 kHz)on an atomically flat cleaved mica (001) surface
immersed in 20 ml of peptide solutions. The typical scan
speed was about 3 min/image.
Figures 3A and B–D show AFM images of the a3peptide at 4 and 50 mM, in phosphate buffer with pHs
5–6. No fibrous structures were observed at pH 2, 7 or 8.
It is clear that the a3-peptide at 50 mM formed fibrils as
demonstrated by TEM, whereas at 4 mM no such fibrousassemblies were observed. Fibrils were observed between
pHs 3 and 6. Each single fibril had a length of >1, 000 nm.
At pH 5.5, each fibril had a width of 33.0  0.4 and2.1  0.4 nm in height.
When the 5Qa3- and 7Qa3-peptides at 40 mM wereobserved by AFM, no fibrous assemblies were observed.
Since the a-helices of these peptides were less stable thanthat of the a3-peptide, as demonstrated by CD measurements, AFM was carried out at higher peptide
J. Biochem.

Requirement of Ala Residues for Fibre Formation

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Fig. 3. AFM images of the a3-peptide from 4 mM solution at pH 5.5 (A) and from 50 mM solution at pHs 5 (B), 5.5 (C) and 6(D). A single fibre has a length of >1000 nm.

Fig. 4. AFM images of the 5Qa3-peptide (A) and 7Qa3-peptide (B) from 400 kM solution.

concentrations. When the concentration of the 5Qa3peptide was increased up to 400 mM, many very shortfibrils with long-elliptical shape were detected, as shownin Fig. 4A. Each single 5Qa3-peptide fibril was 400 
100 nm in length, 2.9  0.8 nm in height and 61.7  8.5 nm
Vol. 144, No. 1, 2008

in width. However, no such structures were observed forthe 7Qa3-peptide at the same peptide concentration(Fig. 4B). These results strongly indicate that Ala residues
at the g position of the heptad sequence of the a3-peptide
largely contribute to the formation of fibrous assemblies.

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G. Aoki et al.

The destabilization of the a-helix formed by the 5Qa3peptide is considered to be due to the difference ina-helix formation tendency between Gln and Ala.
However, since some degree of hydrophobicity is retainedin the 5Qa3-peptide, very short fibrils with long-ellipticalshape of this peptide might have been observed by AFM,
although a higher peptide concentration was required
for such observation. In contrast, the 7Qa3-peptide that
also has Gln residues did not form fibrous assemblies.
To explain this phenomenon, we considered that thechemical environments around the Gln residues in thehelical bundle structure differed between the 5Qa3- and7Qa3-peptides. From the helical wheel representation(Fig. 1), the surface formed by c-, d- and g-position
residues is more hydrophobic than the opposite surface
formed by a-, b- and e-position residues, since a Thr
residue with methyl and hydroxyl groups is morehydrophobic than a Glu residue with a negative charge.
Therefore, the 5Qa3-peptide that retains a more hydrophobic surface is considered to have an ability to form
supramolecular assemblies, whereas the 7Qa3-peptide
in which the hydrophobicity of the surface formed byc-, d- and g-position residues is weakened by Ala!Gln
substitution at the g position seems to lose its ability toform such assemblies. Zeng et al. (35) have previously
produced various e- and g-position mutants of a twostranded GCN4 leucine zipper by random mutagenesis
and have shown that type II mutants that form higher
order oligomers commonly have Ala residues at the gposition. Their finding is consistent with our results inthis study. Therefore, similar mechanisms of forming
higher order oligomers may operate in their peptides andours; however, they provided no explanation for theirfinding.
It is also demonstrated that a decrease in thehydrophobicity of the helical surface formed by c-, dand g-position residues of the a3-peptide by Leu!Val
substitution at the d position results in the loss of
the ability to form fibrous assemblies (33). However,in this case, the resultant peptide (4Va3-peptide) also
had no ability to form a-helices; thus, the formation offibrous assemblies is closely related to a-helix formation.
In contrast, the 7Qa3-peptide in this study lost its ability
to form fibrous assemblies in spite of retaining its ability
for a-helix formation. Therefore, Ala residues at the gposition in the a3-peptide are concluded to be the key
residues for forming fibrous assemblies. In the future,subsequent analyses using a3-derived peptides withvarious sequences will clarify the detailed mechanismsof the fibre formation of the a3-peptide.
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