Spectroscopic and DNA-binding characterization of the
isolated heme-bound basic helix–loop–helix-PAS-A domain
of neuronal PAS protein 2 (NPAS2), a transcription
activator protein associated with circadian rhythms
Yuji Mukaiyama
1
, Takeshi Uchida
2
*, Emiko Sato
1
, Ai Sasaki
1
, Yuko Sato
1
, Jotaro Igarashi
1
,
Hirofumi Kurokawa
1
, Ikuko Sagami
1
†, Teizo Kitagawa
2
and Toru Shimizu
1
1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan
2 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan
In animals, biological rhythms are coordinated in
adaptive synchrony by the brain, specifically by the
suprachiasmatic nucleus of the hypothalamus. The
suprachiasmatic nucleus is a major coordinator of

doi:10.1111/j.1742-4658.2006.05259.x
Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated
transcription factor with two heme-binding sites on two PAS domains. In
the present study, we compared the optical absorption spectra, resonance
Raman spectra, heme-binding kinetics and DNA-binding characteristics of
the isolated fragment containing the N-terminal basic helix–loop–helix
(bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the
PAS-A domain alone. We found that the heme-bound bHLH-PAS-A
domain mainly exists as a dimer in solution. The Soret absorption peak of
the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wave-
length 9 nm higher than for isolated PAS-A (412 nm). The axial ligand
trans to CO in bHLH-PAS-A appears to be His, based on the resonance
Raman spectra. In addition, the rate constant for heme association with
apo-bHLH-PAS (3.3 · 10
7
mol
)1
Æs
)1
) was more than two orders of magni-
tude higher than for association with apo-PAS-A (< 10
5
mol
)1
Æs
)1
). These
results suggest that the bHLH domain assists in stable heme binding to
NPAS2. Both optical and resonance Raman spectra indicated that the
Fe(II)–NO heme complex is five-coordinated. Using the quartz-crystal

N-terminal region, but in contrast to CLOCK, both
the PAS-A and PAS-B domains of NPAS2 contain a
heme-binding site, and CO binding to the heme inhib-
its the DNA-binding activity of the NAPS2–BMAL1
heterodimer [15].
The bHLH-PAS proteins are critical regulators of
gene expression networks underlying a variety of essen-
tial physiological and developmental processes [7,16].
In many cases, bHLH proteins dimerize to form func-
tional DNA-binding complexes, whereas bHLH-PAS
proteins are distinct from other members of the
broader bHLH superfamily due to the dimerization
specificity conferred by their PAS domains. The
bHLH-PAS proteins tend to be ubiquitous latent sig-
nal-regulated transcription factors that often recognize
variant forms of the classic E-box enhancer sequence
bound by other bHLH proteins [7,16]. Because CO
binding to the heme causes dissociation of NPAS2
from BMAL1 and the E-box sequence, the bound
heme itself may affect the DNA-binding properties of
NPAS2 by interacting with the bHLH region in a
direct or indirect manner. Therefore, it is worth study-
ing how the bHLH domain contributes to heme bind-
ing to the PAS-A domain in NPAS2 and how the
bound heme participates in the binding of NPAS-2 to
the E-box DNA sequence.
In the present study, we investigated the role of the
bHLH domain by characterizing the isolated heme-
bound bHLH-PAS-A and heme-bound PAS-A
domains of NPAS2 using optical absorption spectros-

Size exclusion chromatography
Gel filtration analysis of a solution of bHLH-PAS-A
protein using Superdex 75 (Amersham Biosciences,
Uppsala, Sweden) revealed that the solution contains
one major peak (more than 70%) and two minor
peaks (supplementary Fig. S2A). The heme-reconstitu-
ted bHLH-PAS-A domain was the major species and
had a molecular mass of nearly 60 kDa (supplement-
ary Fig. S2B). Because the monomer has a predicted
molecular mass of 28 kDa, this suggests that the major
species of the bHLH-PAS-A domain exists as a dimer.
We collected only the major fraction and reapplied it
to the Superdex 75 column. The chromatographic pro-
file was the same, suggesting that the fraction was in
equilibrium between monomer, dimer (majority) and
trimer forms.
Optical absorption spectra of Fe(III), Fe(II) and
Fe(II)–CO complexes
To understand the heme environment of the bHLH-
PAS-A domain of NPAS2, we obtained optical
absorption spectra of the overexpressed and purified
bHLH-PAS-A domain. Optical absorption spectra of
the Fe(III), Fe(II) and Fe(II)–CO complexes of the
bHLH-PAS-A domain of NPAS2 are shown in
Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2
FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2529
Fig. 1A and are summarized in Table 1. For the
Fe(III) form, the absorption maxima were located at
421 nm and 543 nm at pH 8.0. The absorption max-
ima of the Fe(II) species were at 426, 530 and 559 nm,

3.0
2.0
1.0
0
.
0
Absorbance
00700600
5
0
0
4003
)mn(
h
tgnelev
aW
3×
)III(eF
)II(eF
OC-)II(eF
8.0
6
.0
4
.0
2.0
0.0
Absorbance
0070
0

Visible
(nm)
Fe(III) 421 543 412 538
Fe(II) 426 530, 559 423 530, 558
Fe(II)–CO 422 538, 566 420 530, 568
Intensity
007100610051004100310
0
21
t
fihs
nam
a
R
(mc
1-
)
1640
1579
1504
1631
1471
1359
1493
1555
1618
1605
)III(eF
)II(eF
1582

1493 cm
)1
, representing the five-coordinate high-spin
and six-coordinate low-spin states, respectively.
Because of the sensitivity of the Fe–CO and C–O
stretching frequencies to the heme environment
(i.e. electrostatic and steric interactions with surround-
ing groups), spectra of CO adducts of heme proteins
provide valuable information about the heme pocket
[17,32]. Low-frequency and high-frequency regions of
the resonance Raman spectra of the Fe(II)–CO complex
of the bHLH-PAS-A domain are shown in Fig. 3A,B.
Isotope-sensitive lines were observed at 486 cm
)1
for the
Fe–CO stretching mode (m
Fe–CO
) and at 1919 cm
)1
for
the C–O stretching mode (m
C–O
) when we used
13
C
18
O.
Finally, we assigned the 495 and 1962 cm
)1
bands to

2
(cm
)1
) m
3
(cm
)1
) Coordination
Fe(III) 1552 1492 5cHS
1579 1504 6cLS
Fe(II) 1555 1471 5cHS
1582 1493 6cLS
mc(
1–
)
Intensity
00700
6
0050040
0
3
mc
(tfi
h
Sn
a
m
a
R
1–

1962
1919
1962
B
Fig. 3. Effects of isotopically labeled CO molecules on resonance Raman spectra of the Fe (II)–CO complexes of basic helix–loop–helix
(bHLH)-PAS-A in the low-frequency (A) and high-frequency (B) regions. The bottom lines show the
12
C
16
O complex, the middle lines the
13
C
18
O complex, and the top lines the difference spectra between the
12
C
16
O and
13
C
18
O complexes.
Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2
FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2531
Raman spectra of the Fe(II)–NO complex. As shown
in Fig. 5, the Fe(II)–NO complex had a Soret absorp-
tion peak at 394 nm, which is characteristic of a five-
coordinated Fe(II)–NO heme complex [23–27,32]. In
addition, the characteristics of the resonance Raman
spectra of the Fe(II)–NO complex (Fig. 6) were very

absorbance at 421 and 419 nm and a decrease at
408 nm, which correspond to the binding of Fe(II)–
CO heme to the apo-bHLS-PAS-A domain and the
apo-PAS-A domain, respectively. This shows that
Fe(II)–CO heme bound strongly to the apo-bHLH-
PAS-A domain (Fig. 7A). The time-dependent
increase in absorbance accompanying Fe(II)–CO
heme binding to the apo-bHLH-PAS-A domain was
composed of only a single phase (Fig. 7A, inset),
and the rate of association was dependent on the
apoprotein concentration (Fig. 7C). As summarized
in Table 4, the rate constant for association with the
apo-bHLH-PAS-A domain was 3.3 · 10
7
mol
)1
Æs
)1
.
In contrast, Fe(II)–CO heme binding to the apo-
PAS-A domain was very slow and did not saturate
under our experimental conditions (Fig. 7B). There-
fore, the k
on
value of the PAS-A domain should be
less than 10
5
mol
)1
Æs

of DNA–protein and protein–protein interactions in
solution, which are monitored by the linear decreases
of the emitted frequency with increasing mass present
01.
0
80.0
60.0
40
.
0
2
0.0
0
0.0
Absorbance
00700600500400
3
)mn(htgelevaW
3×
)II(eF
ON-)II(eF
Fig. 5. Optical absorption spectra of the Fe(II)–NO (bold line) and
Fe(II) (thin line) heme complexes of basic helix–loop–helix (bHLH)-
PAS-A. The position of the Soret band for the Fe(II)–NO complex
(394 nm) suggests that it is a five-coordinated NO–heme complex.
Table 3. Resonance Raman spectra of Fe(II)–CO complexes of the
basic helix–loop–helix (bHLH)-PAS-A and PAS-A domains of neuron-
al PAS domain protein 2.
m
Fe–CO

binding to the E-box sequence, we injected heme-free
bHLH-PAS-A onto the E-box-bound sensor chip. In
this case, a decrease in the frequencies was not
observed (Fig. 9B). To confirm that the binding to the
E-box sequence was specific, we examined the binding
of a mutant E-box sequence (wild type: CACGTG:
mutant GACGTC). Essentially no frequency shift was
observed when either the heme-bound or heme-free
domains were applied to the mutant E-box
(Fig. 9C,D). Similarly, the PAS-A domain without the
bHLH domain did not bind to the E-box sequence.
Also, the heme-bound bHLH-PAS-A domain did not
bind to a random DNA sequence (not shown). Collec-
tively, these results show that the bHLH-PAS-A
domain specifically binds to DNA containing an E-box
sequence under the experimental conditions used.
Because the bHLH-PAS-A forms mainly a dimer in
solution (supplementary Fig. 2S), it may bind to the
E-box as a dimer.
Discussion
The findings from the current study suggest that the
bHLH domain assists and stabilizes heme binding by
the isolated bHLH-PAS-A domain of NPAS2. In addi-
tion, specific binding of the isolated bHLH-PAS-A
domain to the E-box was observed only when it was
bound to heme.
The optical absorption spectra revealed that Fe(III),
Fe(II) and Fe(II)–CO complexes of bHLH-PAS-A and
PAS-A were six-coordinate low spin. The Soret peaks
of Fe(III), Fe(II) and Fe(II)–CO complexes of bHLH-

0
06
10
05
100
41
0
031
tfihS
n
amaR(mc
1–
)
1361
1376
1508
1584
1648
1606
1670
1642
Fig. 6. Effects of isotopically labeled NO molecules on resonance Raman spectra of the Fe(II)–NO complexes of basic helix–loop–helix
(bHLH)-PAS-A in low-frequency (A) and high-frequency (B) regions. The bottom lines show the
14
N
16
O complex, the middle lines the
15
N
16

Æs
)1
)
k
off
(s
)1
)
K
d
(M) References
bHLH-PAS-A 3.3 · 10
7
5.3 · 10
)3
1.6 · 10
)10
This work
PAS-A < 10
5
This work
PAS-B
a
7.7 · 10
5
3.2 · 10
)3
[21]
<10
5

)14
[18]
a
Spectral changes observed for both association and dissociation
were composed of two phases.
b
Data for heme-regulated eIF2a
kinase (HRI) were taken from the PhD thesis of J. Igarashi (Tohoku
University, Sendai, Japan).
03.
0
02.0
0
1
.0
Absorbance
0640440
2
400
4
083
)mn(ht
gnele
v
a
W
A
B
62
.0

3
A
0
1
x
5
5
3
-
05
5
4
04
5
3
Absobance at 421 nm
0
.
2
5.
1
0
.
1
5.00
.0
)
s
(
emiT

01
x
0.
2
6
-
5
.1
0.15
.
00
.0
)
M(]
A-S
AP/HLHb[
Fig. 7. Optical absorption spectral changes accompanying assoc-
iation of Fe(II)–CO heme with heme-free basic helix–loop–helix
(bHLH)-PAS-A (A) and PAS-A (B) after mixing using a stopped-flow
spectrometer. The inset in (A) shows the spectral change at
421 nm, which was composed of only a single phase. The correl-
ation between k
obs
and the concentration of heme-free bHLH-
PAS-A is shown in (C).
Characterization of bHLH-PAS-A of NPAS2 Y. Mukaiyama et al.
2534 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS
ted low spin (Table 2). In addition, the inverse correla-
tion between m
Fe–CO

bHLH-PAS-A of NPAS2 was much higher than that
for the isolated PAS-A domain. This further supports
the idea that the bHLH region assists with the stable
binding of heme to the PAS-A domain in the isolated
bHLH-PAS-A protein.
We also determined the rate constant for the dissoci-
ation of heme (k
off
) from the isolated holo-bHLH-PAS-
A domain. We found that the rate constant was similar
to those of other heme proteins (Table 4). On the basis
of these association and dissociation rate constants, we
estimated the heme dissociation equilibrium constant
(K
d
). The apparent K
d
value of heme for the isolated
bHLH-PAS-A domain was much higher than that of
sperm whale myoglobin, but comparable to that of
heme-regulated kinase inhibitor (unpublished observa-
∆
F (Hz)
∆
F (Hz)
∆
F (Hz)
∆
F (Hz)
s)(emiTs)(emiT

005-
0
0
001
0
004
005300030052000
2
0051000
1
2SAP
N
-ol
o
h
2SAP
N
-ol
o
h
00
5
A
S
B
2SA
P
N-oloh
C
00

a
2SAPN-opa
D
Fig. 9. Quartz crystal microbalance (QCM) analyses for the binding of holo (heme-bound)-basic helix–loop–helix (bHLH)-PAS-A to the E-box
sequence (A), apo (heme-free)-bHLH-PAS-A to the E-box sequence (B), holo-bHLH-PAS-A to the mutant E-box sequence (C), and apo-bHLH-
PAS-A to the mutant E-box sequence (D). Aliquots (5 lL) of holo-bHLH-PAS-A (2.55 lgÆlL
)1
) or apo-bHLH-PAS-A (1.80 lgÆlL
)1
) were added
stepwise to 2 mL of buffer bathing the chip, allowing time for the frequency change to stabilize between each step. Addition of the PAS-A
domain lacking the bHLH domain did not change the frequency. The DNA sequence containing the E-box was 5¢-GGGGCGC
CACGTGA
GAGG-3¢, and that containing the mutant E-box was 5¢-GGGGCGC
GACGTCAGAGG-3¢ (E-box regions are underlined).
Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2
FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2535
tions). Because the heme-regulated kinase inhibitor
responds to the heme concentration in cells by switching
the kinase reaction on or off, heme must bind to the pro-
tein reversibly. Therefore, it is possible that in NPAS-2,
heme reversibly binds to the PAS-A domain. Note that
the spectral change accompanying both association and
dissociation of heme for the isolated PAS-B domain of
NPAS2 was composed of two phases (Table 4).
The QCM data demonstrated that the isolated
bHLH-PAS-A domain binds to the E-box DNA
sequence under specific conditions. In contrast, the
bHLH-truncated PAS-A domain did not bind to the
E-box, and the isolated bHLH-PAS-A domain did not

is a mixture of five-coordinate and six-coordinate and
that the protein exists in solution in an equilibrium
between the monomer, dimer and trimer. These factors
may contribute to the complicated kinetic behavior.
Further studies are required to address this issue.
Based on resonance Raman spectral studies of
His119fiAla, His138fiAla, His171fiAla and
Cys170fiAla mutants of the isolated PAS-A domain, it
has been suggested that His119 and Cys170 are the axial
ligands for the Fe(III) complex, whereas His119 and
His171 are the axial ligands for the Fe(II) complex [22].
Note that some sensor proteins, including PAS proteins,
are known to have substantial flexibility [28–31].
In summary, the present study suggests that the
bHLH domain plays an important role in assisting and
stabilizing heme binding to the PAS-A domain in the
isolated bHLH-PAS-A domain of NPAS2. The QCM
data indicated that the isolated bHLH-PAS-A domain
specifically binds to the E-box DNA sequence. Further
studies using both NPAS2 and BMAL1 are needed to
elucidate the mechanism of DNA binding by NPAS2.
Experimental procedures
Materials
Oligonucleotides (18 bp; E-box, random) and 5¢-biotinylated
oligonucleotides (18 bp) were synthesized by the Nippon
Gene Institute (Sendai, Japan). Restriction enzymes and
modification enzymes were purchased from Takara Bio
(Otsu, Japan), Toyobo (Osaka, Japan), New England Bio-
labs (Beverly, MA), and Nippon Roche (Tokyo, Japan).
Other chemicals weer of the highest grade available and were

dithiothreitol, 1 mm phenylmethylsulfonyl fluoride,
2 lgÆmL
)1
aprotinin, 2 lgÆmL
)1
leupeptin, 2 lgÆmL
)1
pepstatin A, 2 mm 2-mercaptoethanol) and lysed by pulse
sonication (three times for 2 min each with 2 min inter-
vals) on ice using an Ultrasonic Disruptor UD-201 (Tomy
Seiko, Tokyo, Japan). Hemin (100 lm final concentration)
dissolved in 0.01 m NaOH was added to this lysate, and
the mixture was allowed to equilibrate on ice for 30 min.
After centrifugation at 35 000 g for 30 min, the superna-
Characterization of bHLH-PAS-A of NPAS2 Y. Mukaiyama et al.
2536 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS
tant was adjusted to 20% saturated ammonium sulfate
and incubated for 30 min on ice. After centrifugation at
35 000 g, the supernatant was adjusted to 70% saturated
ammonium sulfate. Precipitates from the 70% saturated
solution were collected by centrifugation and dissolved in
buffer A. The excess ammonium sulfate was removed and
the buffer was exchanged by applying the solution to
Sephadex G-25 (100 mL) that had been pre-equilibrated
with buffer B (50 mm sodium phosphate (pH 7.8), 50 mm
NaCl, 10% glycerol, 2 mm dithiothreitol). The resulting
solution was applied to an Ni-NTA column (Qiagen, Hil-
den, Germany) pre-equilibrated with buffer C (50 mm
sodium phosphate (pH 7.8), 50 mm NaCl, 2 mm 2-merca-
ptoethanol). The column was washed stepwise with buffer

lar mass and the elution volumes for the following standard
proteins: albumin (67 kDa), ovalbumin (43 kDa), chymo-
trypsinogen A (25 kDa), and ribonuclease A (13.7 kDa).
Optical absorption spectra
Spectral experiments were carried out under aerobic condi-
tions using a Shimadzu UV-2500 spectrophotometer main-
tained at 25 °C with a temperature controller. Anaerobic
spectral experiments were conducted using a Shimadzu UV-
160 A spectrophotometer at 16 °C. When the heme was
reduced by sodium dithionite, the sample solution was sat-
urated with argon gas.
Resonance Raman spectra
The bHLH-PAS-A domain of NPAS2 (35 lm in 100 mm
Tris ⁄ HCl (pH 8.0) and 10% glycerol) was placed in an air-
tight spinning cell with a rubber septum and reduced by the
addition of sodium dithionite (10 mm final concentration).
12
C
16
O,
13
C
18
O
14
N
16
Oor
15
N

the Fe(III) bHLH-PAS-A domain of NPAS2 was examined
as Fe(III) myoglobin formation by monitoring the increase
in absorbance at 410 nm upon mixture of the Fe(III)
bHLH-PAS-A domain of NPAS2 (3 lm) with a 10-fold
excess of H64Y ⁄ V68F apomyoglobin (30 lm) in potassium
phosphate buffer (pH 7.0) containing 0.6 m sucrose at
25 °C [17,18].
CO-binding kinetics
To measure the CO association rates, the bHLH-PASA
domain of NPAS2 (10 lm) was reduced with sodium
dithionite in 50 mm Tris ⁄ HCl (pH 8.0) buffer. This solu-
tion was then rapidly mixed with controlled CO-saturated
buffer (c. 1mm CO) using a stopped-flow spectrophotom-
Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2
FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2537
eter under aerobic conditions at 25 °C. Binding of CO to
the Fe(II) bHLH-PAS-A domain of NPAS2 was monit-
ored at 423 nm. Experiments were performed at least
twice.
Analysis of DNA binding using the QCM
apparatus
Biotinylated DNA (biotin-5¢-AGGGGCGCCACGTGA
GAGGCCT-3¢) was first immobilized to a NewtrAbidinÔ-
bound QCM (AffinixÔQ; Initium Ltd, Tokyo, Japan) [19].
The cleaned Au electrode side of the QCM plate was incu-
bated with NewtrAbidin (1 mg ÆmL
)1
) for 1 h at room tem-
perature and washed with TBS (20 mm Tris ⁄ HCl (pH 7.5),
50 mm NaCl) and fixed to the Affinx

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Supplementary material
The following supplementary material is available
online:
Fig. S1. Sodium dodecyl sulfate-polyacrylamide gel
electorphoresis followed by staining with Coomassie
blue R250.
Fig. S2. Gel filtration chromatography of His-tag-free
bHLH-PAS-A.
This material is available as part of the online article
from
Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2
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