Haem binding and catalytic activity of two-α-helix peptide annealed by trifluoroethanol

Article abstract of DOI:10.1039/a701852e

A designed two-α-helix peptide His-2α binds effectively Fe^III-mesoporphyrin (haem) and enhances N-demethylase activity of the haem, when the 2α-helix structure is annealed by the addition of 10-20% trifluoroethanol.

Full text of DOI:10.1039/a701852e

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Haem binding and catalytic activity of two-a-helix peptide annealed by
trifluoroethanol
Seiji Sakamoto, Shigeo Sakurai, Akihiko Ueno and Hisakazu Mihara*
Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Yokohama
226, Japan
8
9
A designed two-a-helix peptide His-2a binds effectively
stabilizing solvent, the a-helicity of His-2a gradually in-
creased until it reached about 70% at 30% TFE [Fig. 2(b)].
Interestingly, in the presence of the haem, a further increase in
a-helicity was observed at 10–20% TFE [Fig. 2(b), closed
circles]. The largest increase of a-helicity by the addition of
haem was obtained at around 15% TFE (1.4-fold). When His-
2a was titrated with the haem in 15% TFE, the CD spectra of
His-2a changed gradually to that of a typical a-helix structure
with an isodichroic point at 204 nm (Fig. 3). The binding
III
Fe –mesoporphyrin (haem) and enhances N-demethylase
activity of the haem, when the 2a-helix structure is annealed
by the addition of 10–20% trifluoroethanol.
Iron porphyrins occur widely in nature as cofactors of
haemproteins and display diverse functions.1 The specific
function of the haemprotein is determined by the microenvir-
2
onments around the haem. Thus, if we can establish the haem
environments by designing polypeptide 3D structures, it will be
possible to control the haem functions. So far, considerable
effort has been devoted to the conjugation of porphyrin
molecules by chelation or covalent linkage with designed
peptide 3D structures. To develop a mini haemprotein, we here
report the design and synthesis of a 2a-helix peptide His-2a,
which could bind Fe–mesoporphyrin (haem) in a regulated
manner. Furthermore, catalytic activity of the haem that
resembled that of peroxidase was significantly increased by the
binding with the peptide.
constant (K
a
) in 15% TFE, which was estimated from molecular
1
0
ellipticities at 222 nm using a single site binding equation, was
5
3
21
2.8 3 10 dm mol . The addition of 1-methylimidazole (200
equiv.) inhibited the coordination of His-2a with the haem,
resulting in the decrease of the a-helicity to the level without the
haem. Additionally, there was no significant change in the CD
spectra by the addition of haem at acidic pH (2.0–6.0). Because
3
4–6
a
the pK of imidazole is about 6.0, the pH effect is attributed to
the protonation of His side chains such that they cannot act as
ligands. Therefore, we concluded that the increase in a-helicity
A 14-peptide segment in the peptide was designed to take an
amphiphilic a-helix structure in a manner similar to that of a
portion of coiled-coil proteins (Fig. 1).7,8 The two segments
were dimerized by disulfide linkage of the Cys16 residues. As
axial ligands of haem, His residues were introduced at the sixth
position instead of Leu to deploy a haem parallel to the helix.
The peptide was synthesized by the solid-phase method using
the Fmoc strategy and purified with HPLC to high purity
(a)
(b)
100
4
2
80
60
40
5
4
3
2
1
0
0
i
ii
iii
iv
v
(
[
> 98%). The peptide gave a molecular ion peak at m/z 3529.6
(M + H) ] (calc. 3529.1) on matrix assisted laser desorption
+
–2
20
0
0
20
40
TFE (%)
60
ionization times-of-flight mass spectrometry. The monomer
peptide (His-1a) was also synthesized and gave an ion peak at
–
4
+
200
220
240
0
10 20 30 40 50 60
TFE (%)
m/z 1836.9 [(M + H) ] (calc. 1837.2).
The conformation of His-2a (2.0 3 10 mol dm23) in buffer
2
5
λ / nm
(pH 7.4) was almost random due to the introduction of His
Fig. 2 (a) CD spectra of His-2a with increasing percentage volume of TFE
in 2.0 3 1022 mol dm23 Tris HCl buffer (pH 7.4) at 25 °C. i, 0%; ii, 15%;
residues (Fig. 2).† With increasing percentage volume of
trifluoroethanol (TFE), which is known to be an a-helix
2
5
23
iii, 30%; iv, 60%; v, 97%. [His-2a ] = 2.0 3 10 mol dm . (b) Effect of
TFE content on the a-helicity (2) in the absence and (5) the presence of
2
5
23
haem (2.0 3 10 mol dm ). Inset: increase in ellipticity at 222 nm by the
(
(
a) Ac-AlaLeuGluGlnLysHisAlaAlaLeuGluGlnLysLeuAla-βAlaCys-NH2
addition of haem as a function of TFE content.
S
S
(a)
4
2
(b)
Ac-AlaLeuGluGlnLysHisAlaAlaLeuGluGlnLysLeuAla-βAlaCys-NH2
His-2α
S(Acm)
Ac-AlaLeuGluGlnLysHisAlaAlaLeuGluGlnLysLeuAla-βAlaCys-NH2
His-1α
8
b)
[
Haem] / [Peptide]
6
4
2
0
0
Lys12
His-2α
Glu103
0.00
0.26
0.51
0.77
1.03
5.41
Ala8
Lys5
Ala14
9
Glu
1
Leu
Leu
Leu136
Ala7
Ala
2
Gln1
1
His
–2
–4
4
Gln4
11
Gln
His6
Leu
2
His-1α
Leu
Gln
1
7
Ala
13
9
Glu3
Glu
Leu
Lys5
Lys12
Ala
Ala8
14
Ala
10
200
220
λ / nm
240
0
2
4
6
8
10
–
5
–3
[Haem] / 10 mol dm
Fig. 1 Structure of the designed peptides, His-2a and His-1a. (a) Amino
acid sequences of His-2a and His-1a. (b) Illustration of two-a-helix peptide
structure bound to the haem and helix wheel drawing of the two 14-peptides
in coiled-coil form. Acm = acetamidomethyl.
Fig. 3 (a) CD spectra of His-2a with increasing haem concentration in the
5
2
23
buffer containing 15% TFE at 25 °C. [His-2a] = 2.0 3 10
mol dm .
2
5
23
(b) Improvement of a-helix for (2) His-2a (2.0 3 10 mol dm ) and (5)
2
5
23
His-1a (4.0 310 mol dm ) by the addition of the haem.
Chem. Commun., 1997
1221

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of His-2a took place via the haem binding by ligation with His
residues. In contrast, a-helicity of His-1a was little affected by
the addition of haem at any percentage volume of TFE (0–90%),
indicating that His-1a could not bind the haem in this
concentration.
formation of a hydrophobic pocket are important for the haem
binding. That is, at the lower TFE content ( < 10%), the
hydrophobic pocket is not formed, because the conformation of
His-2a is predominantly a random-coil. On the other hand, at
the higher TFE content ( > 30%), the 2a-helix structure is
destroyed so that each a-helix segment is free to move. Because
there is no hydrophobic pocket at either lower or higher TFE
content, the peptide cannot bind the haem effectively. The
monomer peptide His-1a and 1-methylimidazole needed a
Titration of the haem with His-2a was also carried out in the
buffer containing various amounts of TFE. At 15% TFE, the
Soret band at 401 nm increased and the band at around 355 nm
decreased upon the addition of His-2a [Fig. 4(a)]. That is, an
UV–VIS spectrum of the haem was converted from the high
spin to the low spin form with an isosbestic point at 390 nm. The
decrease in absorbance at 355 nm indicated that the peptide
2
3
23
concentration of an order of ca. 10 mol dm for the haem
binding and did not show such a TFE dependence. These results
also confirm that the formation of the 2a-helix structure is
essential for the haem binding.
6
acted to break up haem aggregates. The binding constant for
the haem with His-2a was strongly dependent on the TFE
Next, demethylation catalysis of the haem bound to His-2a
1
1
content [Fig. 4(b)]. His-2a showed the highest binding constant
was demonstrated using N,N-dimethylaniline as a substrate.
5
3
21
at 15% TFE (K
a
= 5.8 3 10 dm mol ). In contrast, His-2a
As shown in Fig. 5, the initial rate v of the reaction in the
2
5
23
21
could not bind the haem effectively at < 10% and > 30% TFE
presence of His-2a (v = 16 3 10 mol dm min ) was
4
3
21
(K
a
< 3.0 3 10 dm mol ). These TFE titrations revealed that
accelerated by a factor of 8.0, relative to that in the absence of
2
5
23
21
the 2a-helix structure annealed by TFE and the consequent
the peptide (v = 2.0 3 10
mol dm
min ). The
acceleration of the reaction was dependent on the TFE content
and showed a maximum value at 15% TFE. The N-demethylase
activity was comparable to that of bilayer-bound cytochrome c
as reported by Hamachi et al.11 The initial rate was dependent
(
a) 1.0
0.6
0.4
[
Peptide] / [Haem]
2.71
2.17
1.62
1.36
1.08
0.81
0.54
0.8
0.6
0.4
0.2
2 2
on the concentration of H O , but not on that of N,N-
0.2
K
5
=
dimethylaniline. Therefore, the rate-determining step is the
reaction between the haem and H O . The peptide seems to
a
.8 × 10 mol–1 dm3
5
2
2
0.0
0
10
20
30
enhance the activity by isolating the haem in the peptide
structure from the haem aggregates in solution.
Peptide] / 10–6 mol dm
–3
[
0.27
.00
0
In conclusion, the haem binding of the His-peptide was
controlled by the peptide conformation with TFE. The catalytic
activity was enhanced by the formation of the haem–peptide
complex, which might be another kind of catalytic molten
× 3
0.0
12
globule. The strategy using designed peptides conjugated with
300
400
500
λ / nm
600
700
functional groups, such as haem, is expected to be applied to the
elucidation of the roles of polypeptide 3D structure on the
diverse functions of natural proteins, and the obtained informa-
tion will be useful for the design artificial proteins.
(b)
6
5
4
3
2
1
0
Footnotes
*
†
E-mail: hmihara@bio.titech.ac.jp
Leu-2a, a non-His peptide, had 55% helicity in the buffer.
K
0
10
20
30
40
50
TFE (%)
References
Fig. 4 (a) UV–VIS spectra of the haem with increasing His-2a
concentration in the buffer containing 15% TFE at 25 °C. [haem] = 1.0 3
1 The Porphyrins, ed. D. Dolphin, Academic, New York, 1979, vol. 7.
2 T. L. Poulos, Adv. Inorg. Biochem., 1988, 7, 2.
3 F. Rabanal, W. F. DeGrado and P. L. Dutton, J. Am. Chem. Soc., 1996,
118, 473.
2
5
23
10
mol dm . (b) Effect of TFE content on the binding constant for His-
2a with the haem.
4
5
T. Sasaki and E. T. Kaiser, J. Am. Chem. Soc., 1989, 111, 380.
H. Mihara, Y. Haruta, S. Sakamoto, N. Nishino and H. Aoyagi, Chem.
Lett., 1996, 1; H. Mihara, K. Tomizaki, T. Fujimoto, S. Sakamoto,
H. Aoyagi and N. Nishino, Chem. Lett., 1996, 187.
Me
H
N
N
Haem
aggregates
Me
Me
+
H2O2
+ HCHO
6
D. R. Benson, B. R. Hart, X. Zhu and M. B. Doughty, J. Am. Chem. Soc.,
1995, 117, 8502.
n
5
4
3
2
1
00
00
00
00
00
0
+
His-2α
Haem + His-2α
7 S. Sakamoto, H. Aoyagi, N. Nakashima and H. Mihara, J. Chem. Soc.,
Perkin Trans. 2, 1996, 2319.
ν = 16 × 10 mol dm–3 min–1
–
5
(8.0)
8
N. E. Zhou, C. M. Kay and R. S. Hodges, J. Biol. Chem., 1992, 267,
664.
J. M. Scholtz, H. Qian, E. J. York, J. M. Stewart and R. L. Baldwin,
2
9
Haem only
Biopolymers, 1991, 31, 1463.
ν = 2.0 × 10 mol dm–3 min–1
–5
1
0 T. Kuwaraba, A. Nakamura, A. Ueno and F. Toda, J. Phys. Chem., 1994,
8, 6297.
11 I. Hamachi, A. Fujita and T. Kunitake, J. Am. Chem. Soc., 1994, 116,
811.
(1.0)
9
0
1
2
3
4
5
8
t / min
1
2 W. F. DeGrado, Nature, 1993, 365, 488; K. Johnsson, R. K. Allemann,
H. Widmer and S. A. Benner, Nature, 1993, 365, 530; H. Mihara,
K. Tomizaki, N. Nishino and T. Fujimoto, Chem. Lett., 1993, 1533;
K. Broo, L. Brive, A.-C. Lundh, P. Ahlberg and L. Baltzer, J. Am. Chem.
Soc., 1996, 118, 8172.
Fig. 5 Time course of formaldehyde formation catalysed by the haem (2)
in the presence and (5) absence of His-2a. The reaction was initiated by
23
23
addition of hydrogen peroxide (4.9 3 10 mol dm ) to mixtures of N,N-
23
23
26
23
dimethylaniline (5.0 3 10 mol dm ), haem (4.8 3 10 mol dm ) and
25
23
23
His-2a (1.0 3 10 mol dm ) in 0.1 mol dm Tris HCl buffer (pH 7.4)
containing 15% TFE at 30 °C. About 80% of the haem was bound to the
peptide under the conditions according to the binding constant.
Received in Cambridge, UK, 17th March 1997; Com.
7/01852E
1222
Chem. Commun., 1997