Báo cáo khoa học: Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms doc

In this study, we overexpressed wild-type and His mutants of the PAS-B domain residues 241–416 of mouse NPAS2 and then purified and characterized the isolated heme-bound proteins.. Optica

heme-bound PAS-B domain of neuronal PAS domain

protein 2 associated with circadian rhythms

Ryoji Koudo1, Hirofumi Kurokawa1, Emiko Sato1, Jotaro Igarashi1, Takeshi Uchida2,

Ikuko Sagami1*, Teizo Kitagawa2and Toru Shimizu1

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

Circadian rhythms are composed of complicated

feed-back loops [1–10] In the suprachiasmatic nucleus, two

proteins, CLOCK and BMAL1, form a heterodimer

that binds to a specific DNA sequence named the

E-box, which is located in the promoter region of

genes associated with clock oscillation, for example

Perand Cry The binding of the CLOCK-BMAL1

het-erodimer to the E-box initiates transcription, and the translated proteins, in turn, negatively regulate the ini-tial transcription, thus constituting a feedback loop A very similar type of feedback regulation is thought to function in the forebrain [11], wherein a transcription protein named neuronal PAS protein 2 (NPAS2), in place of CLOCK, forms a heterodimer with BMAL1

Keywords

circadian rhythms; heme-sensor protein;

PAS domain; resonance Raman

spectroscopy; transcription

Correspondence

T Shimizu, Institute of Multidisciplinary

Research for Advanced Materials, Tohoku

University, 2-1-1 Katahira, Aoba-ku, Sendai

980-8577, Japan

Fax: +81 22 217 5604 ⁄ 5390

Tel: +81 22 217 5604 ⁄ 5605

E-mail: shimizu@tagen.tohoku.ac.jp

*Present address

Graduate School of Agriculture, Kyoto

Prefectural University, Nakaragi-cho 1-5,

Shimogamo, Sakyo-ku, Kyoto 606-8522,

Japan

(Received 24 March 2005, revised 15 June

2005, accepted 20 June 2005)

doi:10.1111/j.1742-4658.2005.04828.x

Neuronal PAS domain protein 2 (NPAS2) is an important transcription factor associated with circadian rhythms This protein forms a heterodimer with BMAL1, which binds to the E-box sequence to mediate circadian rhythm-regulated transcription NPAS2 has two PAS domains with heme-binding sites in the N-terminal portion In this study, we overexpressed wild-type and His mutants of the PAS-B domain (residues 241–416) of mouse NPAS2 and then purified and characterized the isolated heme-bound proteins Optical absorption spectra of the wild-type protein showed that the Fe(III), Fe(II) and Fe(II)–CO complexes are 6-co-ordinated low-spin complexes On the other hand, resonance Raman spectra indicated that both the Fe(III) and Fe(II) complexes contain mixtures of 5-co-ordi-nated high-spin and 6-co-ordi5-co-ordi-nated low-spin complexes Based on inverse correlation between mFe-CO and mC-O of the resonance Raman spectra, it appeared that the axial ligand trans to CO of the heme-bound PAS-B is His Six His mutants (His266Ala, His289Ala, His300Ala, His302Ala, His329Ala, and His335Ala) were generated, and their optical absorption spectra were compared The spectrum of the His335Ala mutant indicated that its Fe(III) complex is the 5-co-ordinated high-spin complex, whereas, like the wild-type, the complexes for the five other His mutants were 6-co-ordinated low-spin complexes Thus, our results suggest that one of the axial ligands of Fe(III) in PAS-B is His335 Also, binding kinetics sug-gest that heme binding to the PAS-B domain of NPAS2 is relatively weak compared with that of sperm whale myoglobin

Abbreviations

BJ FixL, a heme-binding oxygen sensor kinase, FixL, from Bradyrhizobium japonicum; HIF2a, hypoxia-inducible factor 2a; NPAS2, neuronal PAS domain protein 2; PAS, acronym formed from the names of Drosophila-period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT) and Drosophila single-minded protein (SIM).

The NPAS2–BMAL1 heterodimer binds to the E-box

and initiates transcription associated with circadian

rhythms A mouse knockout of NPAS2 adapts poorly

to restricted feeding and becomes severely ill [12]

NPAS2 is an 816-amino-acid protein composed of

a basic–helix–loop–helix (bHLH) domain (residues

1–77), two heme-bound PAS domains, PAS-A

(resi-dues 78–240) and PAS-B (resi(resi-dues 241–354), and a

C-terminal nuclear receptor association region

[resi-dues 665–680 for human NPAS2 (or MOP4)] [13]

Both the PAS-A and PAS-B domains contain

heme-binding sites [13] Transcription caused by heme-binding of

the NPAS2–BMAL1 heterodimer is hampered by CO,

an axial heme ligand [13] Therefore, it is likely that

CO binding to the heme in NPAS2 may impair

het-erodimer formation or its DNA binding In addition,

heme biosynthesis has been reported to correlate with

the circadian clock system [15] However, as NPAS2

has two heme-binding sites, it is unclear which heme

contributes more to the binding of CO and the

inhibi-tion of transcripinhibi-tion Also, the heme environment,

including the axial ligands and heme-binding

proper-ties of the apo-NPAS2 protein, is not known

In this study, we attempted to characterize the

heme-binding environment of the isolated PAS-B

domain of NPAS2 Four PAS-B domains with

differ-ent sequences were expressed in Escherichia coli The

most stable PAS-B domain binds one molecule of

heme per molecule of protein, but the domain

appeared to form a large multimer (larger than a

hexa-mer) in both the presence and absence of heme

Opti-cal absorption spectra of both Fe(III) and Fe(II)

complexes indicate that both are 6-co-ordinated

low-spin complexes Based on Raman spectra, one of the

axial ligands appeared to be His Site-directed

muta-genesis indicated that His335 is one of the axial ligands

for heme in the isolated PAS-B protein

Results

Protein expression and purification

We attempted to express four types of the PAS-B

domain with different lengths corresponding to

amino-acid residues 160–346, 218–416, 218–346, and 241–416

Only the PAS-B domain with residues 241–416 was

properly overexpressed and folded in E coli and

dis-played sufficient heme-binding affinity Therefore, we

used the protein consisting of residues 241–416 for

further studies on the structure and function of the

PAS-B domain

His-tagged PAS-B was expressed in E coli cells and

purified as a heme-bound protein in the presence of

hemin It was then treated with thrombin to eliminate the His tag Undigested His-tagged protein was removed by Ni⁄ nitrilotriacetate ⁄ agarose column chro-matography The yield of PAS-B protein was 1.6 mgÆL)1 culture Heme-free PAS-B was also expressed and purified in the absence of hemin Titration experi-ments with hemin demonstrated that purified PAS-B has one binding site per protein with a heme to protein ratio of 1 : 0.91 We estimated the spectral dissociation constant using concentrations of free heme in the solu-tion to be  2 lm (Fig S1 in Supplementary material) Quantification of heme by the pyridine hemochromo-gen method [16] confirmed that heme-saturated PAS-B contains 1.23 hemes per monomer

Optical absorption spectra Figure 1A shows the optical absorption spectra of Fe(III), Fe(II), and Fe(II)–CO complexes of heme-bound wild-type PAS-B Optical absorption spectra of

a His335Ala mutant are also shown in Fig 1B, which

A

B

Fig 1 Optical absorption spectra of Fe(III) (bold solid lines), Fe(II) (thin solid lines), and Fe(II)–CO (broken lines) complexes of the wild-type (A) and His335Ala mutant (B) proteins Spectra were obtained in 100 m M Tris ⁄ HCl (pH 7.5) Spectra of the His266Ala, His289Ala, His300Ala, His329Ala mutants were essentially the same as that of the wild-type protein.

will be discussed in the last part of Results Table 1

summarizes the spectral maxima of the wild-type and

His mutant PAS-B complexes The Soret peaks of the

Fe(III) and Fe(II) complexes of wild-type PAS-B are

located at 419 and 424 nm, respectively Comparison

with other 6-co-ordinated low-spin heme proteins

sug-gested that Fe(III), Fe(II), and Fe(II)–CO wild-type

PAS-B complexes are also 6-co-ordinated low-spin

complexes In addition, the results implied that His is

probably one of the axial ligands of wild-type PAS-B

Resonance Raman spectra

To further understand the heme environment of

PAS-B, resonance Raman spectra were obtained and

com-pared with those of other hemoproteins Resonance

Raman spectra of wild-type PAS-B heme complexes in

the high-frequency region are shown in Fig 2 and

sum-marized in Table 2 Bands at 1373 and 1359 cm)1 for

the Fe(III) and Fe(II) complexes of PAS-B, respectively,

were assigned to redox-sensitive m4 In the Fe(III)

PAS-B complex, the spin-state and co-ordination-state

mar-ker band, m3, was located at 1468 and 1502 cm)1,

suggesting the presence of 6-co-ordinated high-spin and

low-spin complexes, respectively [21] Two m3 bands

observed at 1470 and 1492 cm)1 for the Fe(II) PAS-B

complex represent 5-co-ordinated high-spin and

6-co-ordinated low-spin complexes, respectively Upon

bind-ing of CO to the Fe(II) complex, the m3band shifted to

1498 cm)1, and the presence of a band at 1467 cm)1

indicated partial photodissociation of CO from heme

The CO-isotope dependences of the resonance

Raman spectra of the Fe(II)–CO complex of PAS-B

are shown in Fig 3 The lower and middle spectra in the 300–700 cm)1 region in the left panel and the 1700–2000 cm)1 region in the right panel represent

12CO16O-PAS-B and 13C18O-PAS-B, respectively, whereas the upper spectra in both panels show the

Table 1 Optical absorption spectra (nm) of the wild-type and His mutant proteins of the isolated heme-bound PAS-B domain of NPAS2 Cyt

b5, Cytochrome b5; Cyt b562, cytochrome b562; Sw Mb, sperm whale metmyoglobin.

Proteins

Reference

(His ⁄ His 6cLS)

(His ⁄ His 6cLS)

Cyt b 562 418

(His ⁄ Met 6cLS)

(His ⁄ Met6 cLS)

(His ⁄ H 2 O 6cLS)

(His 5cHS)

556 (His ⁄ CO 6cLS)

Fig 2 Resonance Raman spectra (excited at 413 cm)1) of Fe(III) (bottom), Fe(II) (middle), and Fe(II)-CO (top) complexes of PAS-B.

isotope difference It is clear from the difference

spec-tra that the 497-cm)1 and 1961-cm)1bands of12C16

O-PAS-B are shifted to 489 and 1864 cm)1, respectively,

upon binding of13C18O Accordingly, we assigned the

497-cm)1and 1961-cm)1bands to the mFe-CO and mC-O

modes, respectively In Fig 3 (left panel), the Fe-C-O

bending mode, mFe-C-O, was also identified at 577 cm)1

Figure 4 shows inverse correlations between mFe-C

and mC-O observed in the resonance Raman spectra

The plot for PAS-B falls on the line of the

histidine-co-ordinated heme proteins, indicating a His-Fe-CO

6-co-ordinated adduct with PAS-B

Heme association and dissociation

As shown in Fig 5A, we obtained the association rate

constant for binding of the Fe(II)–CO heme complex

to apo-PAS-B The spectral change monitored at

420 nm was composed of fast (73%) and slow (27%) phases (Fig 5B) The rate constant of the fast phase for the heme association was dependent on the apo-protein concentration (Fig 5C), and the rate constant

of even the fast phase, 7.7· 105m)1Æs)1, was lower than those of other heme-binding proteins (Table 3) Note that SOUL and p22HBP are tentatively consid-ered to be heme-transporting proteins, but their phy-siological roles are not yet certain

We also determined the dissociation rate of heme from Fe(III) PAS-B by adding an excess of the His64-Tyr⁄ Val68Phe apomyoglobin mutant [18] The forma-tion of Fe(III)-bound myoglobin was monitored by following the increase in A410 (Fig 6) The spectral change was also composed of fast (32%) and slow (68%) phases The rate of the major slow change, 3.0· 10)4s)1, is one order of magnitude lower than those of other hemoproteins (Table 3)

Site-directed mutagenesis His is probably the ligand trans to CO in the PAS-B Fe(II)–CO complex To identify which His is the axial ligand, we examined the conserved His residues in sequence alignments of PAS-B and PAS-A of NPAS2,

Table 2 Resonance Raman spectra of Fe(III) and Fe(II) complexes

of PAS-B.

Fig 3 Resonance Raman spectra (excited at 413 cm)1) of the Fe(II)–CO complex of isolated PAS-B in the low (left) and high (right) fre-quency regions The lower spectra are of the Fe(II)– 12 C 16 O complexes, the middle spectra are of the Fe(II)– 13 C 18 O complexes, and the upper spectra are difference spectra between those of the12C16O and13C18O complexes.

PAS-B of hypoxia-inducible factor 2a (HIF2a), and

the heme-binding PAS domains of BJ FixL and Ec

DOS (Fig S2 in Supplementary material) However,

the sequence alignments indicated that, except for

His302 in PAS-B of NPAS2, the His residues are not

well conserved in the heme-binding PAS domains

Optical absorption and resonance Raman spectral

ana-lyses suggested that PAS-B Fe(III) is a 6-co-ordinated

complex similar to PAS-A In PAS-A NPAS2, His119

and Cys170 should be the axial ligands of the Fe(III)

complex, whereas His119 and His171 should be the

axial ligands for the Fe(II) complex [31] We examined

the effects of mutating six His residues in PAS-A

cor-responding to those between His119 and His171 and

those spanning the Hb-sheet Thus, we generated the

following mutants: His335Ala, His266Ala, His289Ala,

His300Ala, His302Ala, and His329Ala As summarized

in Table 1, only the His335Ala mutant had an optical

absorption spectrum different from the wild-type For

example, the spectrum of the Fe(III) His302Ala

mutant (data not shown) was essentially the same as

that of the Fe(III) wild-type (Fig 1A), whereas that of

the Fe(III) His335Ala mutant (Fig 1B) was distinct

and indicated a 5-co-ordinated high-spin Fe(III)

com-plex Therefore, the results suggest that His335 is one

of the axial ligands of heme for both the Fe(III) and

Fe(II) PAS-B proteins

Discussion

In this study, we characterized the isolated

heme-bound PAS-B domain of NPAS2 and identified His335

as one of the axial ligands We found that the isolated

PAS-B domain from NPAS2 forms a large multimer

(larger than a hexamer; see Experimental procedures),

even though SDS⁄ PAGE indicates that the protein is

more than 95% homogeneous (data not shown)

Although the PAS-B domain of the intact NPAS2 pro-tein may play a critical role in heterodimer formation with BMALl, the multimer found in this study appears

to be an artifact due to the truncation Nevertheless, optical absorption spectra of the isolated PAS-B domain indicate one species for the Fe(III), Fe(II), and Fe(II)–CO complexes Resonance Raman spectra indi-cate an apparent mixture of 5-co-ordinated high-spin

A

B

C

Fig 5 Changes in the optical absorption spectra accompanying the association of CO-heme (0.5 l M ) with heme-free PAS-B (3 l M ) upon mixing in a stopped-flow spectrometer (A) The spectral chan-ges at 420 nm are shown in (B), and the correlation between kobs and the concentration of isolated heme-free PAS-B is shown in (C) The spectral change was composed of fast (73%) and slow (27%) phases, and the kobsvalues were taken from the fast phase Fig 4 Inverse correlations between mFe-C vs mC-O in resonance

Raman spectra.

and 6-co-ordinated low-spin complexes in the Fe(III)

and Fe(II) states Two phases were found for heme

association and dissociation processes, which may

reflect heterogeneity of the oligomeric states

Dioum et al [14] first reported heme-binding and

spectroscopic characteristics of the isolated PAS-B

domain as well as those of the bHLH-PAS-A and

bHLH-PAS-A-PAS-B domains They found that absorption peaks of Fe(III) PAS-B are located at 426,

530 and 561 nm, and those of Fe(II)–CO PAS-B are located at 426, 530 and 561 nm These spectral max-ima are different from those that we found (Table 1) This discrepancy may be due, in part, to differences in the method used to prepare the isolated domains Dioum et al prepared the isolated domains by dena-turation of an insoluble suspension of E coli cells overexpressing the protein and then subsequently refolding the protein in the presence of heme In con-trast, our method does not use denaturation–refolding procedures Furthermore, our PAS-B protein contains amino acids 241–416, whereas that of Dioum et al is composed of residues 160–346, which contain part of the PAS-A domain and lack part of the C-terminus of PAS-B (Fig 7) These differences in protein prepar-ation and domain architecture may result in differences

in spectroscopic parameters We also attempted to overexpress and isolate PAS-B with different amino-acid residues (i.e residues 160–346, 218–416, or 218– 346; Fig 7), but we found that they had very low heme-binding affinities, were much less stable, and had lower expression than the PAS-B protein containing amino acids 241–416 (our unpublished observations) Recent crystal and solution structures of PAS proteins and analysis sequence homologies have shown that residues between amino acids 347 and 354 of mouse NPAS2, which were absent in the studies by Dioum

et al make up part of the core of the PAS-B domain [27,28] Therefore, the lack of these residues in the study by Dioum et al may have led to instability of their protein [14]

Dioum et al [14] also examined heme dissociation from the bHLH-PAS-A-PAS-B domain However, because the protein they examined has two heme-bind-ing sites, it is difficult to obtain independent values for

Table 3 Association and dissociation rate constants for heme

bind-ing to the isolated PAS-B domain and other heme proteins Note

that k on values were obtained for the Fe(II)–CO complex, whereas

koff values were obtained for the Fe(III) complex SOUL, a

hexa-meric heme-binding protein expressed in the retina and pineal

gland; p22HBP, a heme-binding protein isolated from mammalian

liver; Sw Mb, sperm whale metmyoglobin.

Proteins k on ( M )1Æs)1) k

off (s)1) Reference PAS-B 7.7 · 10 5

(fast: 73%)

3.2 · 10)3 (fast: 32%)

This work

< 10 5

(slow: 27%)

3.0 · 10)4 (slow: 68%))

6.1 · 10)3 [21]

A

B

Fig 6 Changes in the optical absorption spectra accompanying the

dissociation of heme from isolated PAS-B and association with the

H64Y ⁄ V68F apomyoglobin mutant (A) The formation of

Fe(II)–myo-globin was monitored by measuring the increase in A 410 in the

mix-ture of Fe(III)–PAS-B and H64Y ⁄ V68F apomyoglobin (B).

Fig 7 Constructs of PAS-B domains generated in this study The PAS-B construct containing residues 241–416 had appropriate heme-binding affinity, whereas the PAS-B constructs consisting of amino acids 160–346, 218–416, and 218–346 had low heme-binding affinity and were much less stable than PAS-B containing residues 241–416.

heme binding to each PAS domain The present study

is the first report of heme binding and dissociation

kinetic values of the isolated PAS-B domain

Recent structural studies of heme-bound PAS

pro-teins suggest that ligand binding or a change in the

heme redox state causes profound structural changes

in the heme environment [22,23] These structural

changes in the heme-bound PAS domain are

trans-mitted to the functional domain to regulate catalysis

or DNA binding for transcription Structural changes

in the PAS domain of phototropins induced by light

have also been reported [24,25] PAS domains are

probably very flexible in solution and change their

conformation in response to external signals, allowing

them to transmit the signal to downstream transducer

proteins [26]

Interestingly, it has been suggested that PAS-A, a

heme-binding PAS domain in NPAS2, has His119 and

Cys170 as axial ligands in the Fe(III) complex and that

ligand switching occurs from Cys170 to His171 upon

the reduction of heme [31] Therefore, it is possible

that axial ligand switching of the heme in PAS-B also

occurs upon a change in redox state [23] because the

His335 mutation affected the Fe(III) form but not the

Fe(II) form Because Fe(II)–CO heme was used to

evaluate heme binding and Fe(III) heme was used to

study heme dissociation, the large discrepancy between

the heme-binding rate constant ( 3 s)1) and the heme

dissociation rate constant (3· 10)4s)1; Table 3)

sug-gest that axial ligand switching may occur upon a

change in redox state Also, His residues that we did

not examine by site-directed mutagenesis could be

additional axial ligands for heme in PAS-B

His300 of PAS-B in NPAS2 is conserved in the PAS

proteins examined (Fig S2, Supplementary material)

However, the corresponding His of Ec DOS, His83, is

not the axial ligand; rather, the axial ligand is His77

Therefore, it is possible that co-ordination of heme by

PAS-B and PAS-A of NPAS2 is different from that in

EcDOS

Based on amino-acid alignments (Fig S2,

Supple-mentary material), it appears that His335 of PAS-B in

NPAS2 is located at a turn between the Hb- and

Ib-sheets This His is not conserved in the

heme-bind-ing PAS proteins If the PAS-B domain functions only

in the heme binding, then it can be speculated that the

regulation of transcription by NPAS2 may be

modula-ted not only by CO binding but also by the redox state

of the heme iron In this regard, structural changes

dependent on oxygen binding were observed at the

same turn between the Hb-sheets and Ib-sheets of Ec

DOS [32] For BJ FixL, it has been suggested that CO

binding regulates intermolecular interactions through

the Hb and Ib sheets [33] NMR analysis of PAS-B of HIF2a has also suggested that the Hb-sheets and Ib-sheets interact with ARNT in heterodimer forma-tion [28] Therefore, the turn between Hb-sheets and Ib-sheets appears to play a critical role in various func-tions of PAS proteins, including the interaction of NPAS2 with BMAL1

The PAS domain is critical for the ability of PAS proteins to bind other proteins [27,28] The small chemical compound-binding sites and the flexibility of the PAS domain have been examined by NMR spectr-oscopy [29,30] However, the specific role of the PAS-B domain of NPAS2 remains elusive In separate studies,

we have found that the heme-binding affinity of the isolated PAS-A domain is much higher than that of the isolated PAS-B domain (our unpublished results)

We speculate therefore that the heme in the PAS-A domain plays a more important role than the PAS-B domain in the regulation of transcription by CO In addition, the possible redox-dependent ligand switch-ing of the PAS-B domain could modulate heterodimer formation with BMAL1, leading to altered transcrip-tion

Although CO binding to the NPAS2–BMAL1 hetero-dimer abolishes its ability to promote transcription, the binding of CN, NO, or O2 to the heterodimer could promote heme-dependent transcription, but whe-ther PAS-B is involved in the regulation of transcrip-tion by these ligands remains to be determined Further studies are needed to address the specific role of the hemes in PAS-B and PAS-B in heterodimer formation with BMAL1, DNA binding, and transcrip-tional inhibition by CO [14]

Experimental procedures

Materials Mouse brain was obtained from C57BL⁄ 6 mice An mRNA purification kit was purchased from Amersham Biosciences (Uppsala, Sweden), and an RT-PCR kit was purchased from Roche Diagostics Japan (Tokyo, Japan) Oligonucleo-tides were synthesized at the Nihon Gene Research Labor-atory (Sendai, Japan) The cloning vector, pBluescript SK II(+), was purchased from Toyobo (Osaka, Japan), and the expression vector, pET28a(+), was from Novagen (Darmstadt, Germany) E coli competent cells XL1-blue for cloning were purchased from Novagen, and BL21-CodonPlus(DE3)-RIL cells for protein expression were from Stratagene (La Jolla, CA, USA) Restriction and modifying enzymes for DNA recombination were pur-chased from Takara Shuzo Co (Otsu, Japan), Toyobo, New England Biolabs (Beverly, MA, USA), and Roche

Diagnostics Japan All other chemicals were purchased

from Wako Pure Chemicals (Osaka, Japan)

Construction of NPAS2 PAS-B expression

plasmid

The His-tagged NPAS2 PAS-B expression plasmid was

cre-ated by subcloning into the pET28a(+) expression vector

The cDNAs encoding the full-length mouse NPAS2 PAS-B

domain (residues 241–416) was generated by RT-PCR using

mouse brain RNA The primers for RT-PCR were

CGGGATCCCATATGTGTGTTAGCTGACG-3¢ and

5¢-ACGCGTCGACTTAGTGGGAACTCCTTGAG-3¢ The

sequences of the products were confirmed using a

DSQ-2000 L automatic sequencer (Shimadzu Co., Kyoto, Japan)

and subcloned into the NdeI and SalI sites of the E coli

expression vector, pET28a(+), which introduces a His6tag

at the N-terminus of expressed proteins

To create the His266Ala, His289Ala, His300Ala,

His302Ala, His329Ala, and His335Ala mutants of NPAS2

PAS-B, PCR-based mutagenesis was performed using the

QuikChange mutagenesis kit from Stratagene using

pET28a(+) containing wild-type NPAS2 PAS-B cDNA as

a template The primers used for mutations were as

fol-lows:

5¢-ATTTCTGGATGCCAGAGCTCCTCCAATC-3¢ and

5¢-GATTGGAGGAGCTCTGGCATCCAGAAAT-3¢ for

the His266Ala mutant, 5¢-GGCTACGACTACTACGC

CATTGATGACC-3¢ and 5¢-GGTCATCAATGGCGTAG

TAGTCGTAGCC-3¢ for the His289Ala mutant, 5¢-CTGG

CCAGGTGCGCCCAGCATCTGATG-3¢ and 5¢-CATCA

GATGCTGGGCGCACCTGGCCAG-3¢ for the His300Ala

mutant, 5¢-GTGCCACCAGGCTCTGATGCAGTTTGG-3¢

and 5¢-CCAAACTGCATCAGAGCCTGGTGGCAC-3¢ for

the His302Ala mutant, 5¢-GGTTGCAAACCGCCTACTA

CATCACCTAC-3¢ and 5¢-GTAGGTGATGTAGTAGGC

GGTTTGCAACC-3¢ for the His329Ala mutant, and

CTACATCACCTACGCCCAATGGAACTCC-3¢ and

5¢-GGAGTTCCATTGGGCGTAGGTGATGTAG-3¢ for the

His335Ala mutant

The insertion of these mutations was confirmed by

sequencing

Protein expression and purification

His-tagged NPAS2 PAS-B was expressed in E coli

BL21-CodonPlus(DE3)-RIL cells When the A600 reached 0.6,

protein expression was induced by adjusting the culture to

0.05 mm isopropyl b-d-thiogalactopyranoside and

incuba-ting for 20–24 h The E coil cells expressing NPAS2 PAS-B

were suspended in buffer A (50 mm sodium phosphate,

pH 7.8, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol,

1 mm phenylmethanesulfonyl fluoride, 2 lgÆmL)1aprotinin,

2 lgÆmL)1 leupeptin, 2 lgÆmL)1 pepstatin, and 2 mm

2-mercaptoethanol) and were crushed by pulsed sonication

for 2 min (three times with a 2-min interval between each pulse) on ice using a UD-201 ultrasonic disruptor (Tomy Seiko, Tokyo, Japan) Apo-PAS-B in crushed cells was reconstituted with heme by incubating it with buffer A

con-taining 100 lm heme After centrifugation at 142 000 g for

35 min at 4
C, ammonium sulfate was added to the result-ing supernatant up to 50% saturation Precipitates were collected and then dissolved in buffer A The solution was passed through a Sephadex G-25 column (4· 20 cm) pre-equilibrated with buffer A The eluted solution was applied

to a Ni⁄ nitrilotriacetate ⁄ agarose column pre-equilibrated with buffer B (50 mm sodium phosphate, pH 7.8, 50 mm NaCl, and 2 mm 2-mercaptoethanol) The column was washed with sequential steps of buffer B containing 20 and

50 mm imidazole The NPAS2 PAS-B protein was then eluted with buffer A containing 100 mm imidazole The protein fractions were pooled and concentrated The buffer

in the collected protein was exchanged with 100 mm Tris⁄ HCl (pH 8.0) using a HiTrap desalting column (Amer-sham Biosciences) The purified protein was quickly frozen

in liquid nitrogen and stored at )80
C Protein concen-trations were determined using the CBB dye binding method (Nacalai Tesque, Kyoto, Japan), and heme content was determined by the pyridine hemochromogen method [16]

Removal of His tag by digestion with thrombin His-tagged NPAS2 PAS-B was incubated with 2 eq heme for 3 h at 0
C in 100 mm Tris ⁄ HCl buffer (pH 8.0) Excess heme was then removed using a Bio-Gel P-6 column (Bio-Rad, Hercules, CA, USA) under the same buffer con-ditions Thrombin protease [10 UÆ(mg NPAS2 PAS-B protein))1] was added to the heme-saturated His-tagged NPAS2 PAS-B and incubated for 4 h at 16
C The solu-tion was then applied to a Ni⁄ nitrilotriacetate column pre-equilibrated with 100 mm Tris⁄ HCl (pH 8.0) The flow-through fractions were collected as purified NPAS2 PAS-B lacking the His tag

Size exclusion column chromatography

We used size-exclusion chromatography, Superdex 75, for evaluation of the oligomeric state of the purified protein Apparent molecular masses determined for both heme-bound and heme-free PAS-B proteins were higher than 120 kDa

Optical absorption spectra Optical absorption spectra were collected under aerobic conditions using a Shimadzu UV-2500 and a Shimadzu Multi Spec 1500 spectrophotometer maintained at 25
C

To ensure that the temperature of the solution was appro-priate, the reaction mixture was incubated for 10 min

before spectroscopic measurements Experiments were

per-formed at least three times for each complex

Heme quantification

To quantify heme, 10 lm protein was treated with 30%

(v⁄ v) pyridine in 0.1 m NaOH, after which a few grains of

sodium dithionite were added The heme concentration was

calculated from the difference in A556 and A540 and using

an absorption coefficient of 22.1 mm)1Æcm)1[16]

Resonance Raman measurements

The Fe(III)–PAS-B complex (25 lm in 100 mm Tris⁄ HCl,

pH 8.0) was placed in an airtight spinning cell with a

rubber septum and reduced by the addition of sodium

dithionite (10 mm final concentration) 12C16O or 13C18O

(Cambridge Isotope Laboratories, Andover, MA, USA) gas

was introduced into the Raman cell with an airtight

syr-inge Raman scattering was excited at 413.1 nm with a Kr

ion laser (BeamLok 2060; Spectra-Physics, Mountain View,

CA, USA) The excitation light was focused into the cell at

a laser power of 5 mW for the Fe(III) and Fe(II)

com-plexes For the Fe(II)–CO complexes, to avoid photolysis,

the laser power was 0.1–0.2 mW Raman spectra were

detected with a N2-cooled CCD camera (Spec10: 400BLN;

Roper Scientific, Inc., Trenton, NJ, USA) attached to a

sin-gle polychromator (SPEX750M; Jobin Yvon, Longjumeau,

France) Raman shifts were calibrated with indene, acetone,

CCl4, and an aqueous solution of ferrocyanide

Stopped-flow measurements

Stopped-flow absorbance measurements for obtaining heme

association rate constants were conducted using an

RSP-1000 stopped-flow apparatus (Unisoku, Osaka, Japan)

maintained at 25
C Association of CO-heme with

heme-free PAS-B was monitored at 420 nm The reaction was

ini-tiated by mixing with excess apoprotein Data were fitted

using the data analysis program Igor-Pro (Wavemetrics,

Inc., Oswego, OR, USA) [17]

Heme dissociation experiments

All heme dissociation experiments were carried out in

1-cm path length, 1-mL volume cuvettes containing

800 lL of reaction mixture at 25
C For most

experi-ments, this mixture consisted of 0.15 m potassium

phos-phate (pH 7.0), 0.45 m sucrose, 30 lm apomyoglobin

His64Tyr⁄ Val68Phe, and 3.0 lm stock PAS-B holoprotein

solution The pH values of these reaction mixtures never

deviated more than 0.02 pH unit from that of the original

solution The reactions were initiated by adding

holopro-tein to the buffer⁄ apomyoglobin mixture When multiple

reactions were carried out, absorbance time course data were collected at a single wavelength (410 nm) as des-cribed previously [18]

Acknowledgements

This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Sci-ence and Technology of Japan (to T S and H K.)

We thank Dr John S Olson (Rice University) for kindly providing the expression plasmid for His64-Tyr⁄ Val64Phe apomyoglobin

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Supplementary material

The following material is available online

Fig S1 Spectral changes caused by adding heme to apo-PAS-B The spectral change was composed of two phases It is difficult to estimate the exact dissociation constants from the two phases We tentatively estima-ted the apparent spectral dissociation constant to be

 2 lm

Fig S2 Sequence alignments of PAS-B and PAS-A of NPAS2, PAS-B of HIF2a, a heme-binding PAS domain of BJ FixL and PAS-A of Ec DOS Amino acids at 266, 289, 300, 302, 329, and 335 with bold H

of PAS-B of NPAS2 were mutated to Ala in this study The secondary structure is based on the solution structure of HIF2a PAS-B domain [28]