Supporting information
Materials. Synthetic genes encoding PCNA1, PCNA2 and PCNA3 were purchased from
GenScript (Piscataway, NJ, USA). pHSG398 was purchased from Takara Bio (Shiga, Japan).
pET-15b(+), pET-28b(+) and pLysS were purchased from Novagen (Darmstadt, Germany).
Anhydrotetracycline and pASK-IBA3 plus were purchased from IBA (Goettingen, Germany).
pQE80L-Kan was purchased from Qiagen (Hilden, Germany). pBAD202/D/lacZ was purchased
from Invitrogen (Carlsbad, CA, USA). T7 polynucleotide kinase, T4 DNA ligase and
Escherichia coli T7 Express Iq were purchased from New England Biolabs (Ipswich, MA, USA).
In-Fusion enzyme was purchased from Clontech Laboratories (Mountain View, CA, USA).
Ampicillin, kanamycin, isopropyl β-D-1-thiogalactopyranoside (IPTG) and arabinose were
purchased from Wako Pure Chemical Industries (Osaka, Japan). 5-Aminolevulinic acid
hydrochloride (ALA) was purchased from COSMO BIO (Tokyo, Japan). HisTrap FF crude
column (1.6×2.5 cm), HiTrap Q FF column (1.6×2.5 cm), HiTrap DEAE FF column (1.6×2.5
cm), HiLoad 16/600 Superdex 75 pg column (1.6×60 cm) and Superdex 200 10/300 GL (1.0×30
cm) were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). DE52
preswollen microgranular DEAE cellulose was purchased from Whatman (Maidstone, Kent,
UK).
Vector Construction. The gene encoding PCNA1 was amplified by PCR using two primers
5’-GGAATTCATATGTTTAAAATTGTGTATCCGAACGCC-3’ (forward) and 5’-CGGGATCC
GCTCTTCACAGGCGCGGCGCAATCC-3’ (reverse). The generated DNA fragment was cloned
into pHSG398 between EcoRI and BamHI sites. After the resulting plasmid was digested with
1
NdeI and BamHI, the generated fragment was ligated into pET-15b(+) that was digested with the
same restriction enzymes. The resulting plasmid, pET15b+PCNA1WT, expresses PCNA1WT. The
G108C mutation was introduced by PCR using two primers 5’-AAATCTTGCGCCAAAAGTAC
CATCTAC-3’ (forward) and 5’-TTTGGCGCAAGATTTTTCATCACGGAT-3’ (reverse). The
P186C mutation was introduced by PCR using two primers 5’-GATAAATGCCTGAAAGAACT
GAGCATC-3’ (forward) and 5’-TTTCAGGCATTTATCTT TCATCAGAAA-3’ (reverse).
The gene encoding PCNA2 was amplified by PCR using two primers 5’-GGAATTCAT
ATGATGAAAGCCAAAGTGATCGATG-3’ (forward) and 5’-CGGGATCCGCTCTTCAATCC
GCGCGCGGTGC-3’ (reverse). The generated DNA fragment was digested with NdeI and
BamHI and ligated into pET-15b(+) that was digested with the same restriction enzymes. The
resulting plasmid, pET15b+PCNA2WT, expresses PCNA2WT. The E105C mutation was
introduced by PCR using two primers 5’-GATGGTTGCTTTACCCGCAGTTTTGAA-3’
(forward) and 5’-GGTAAAGCAACCATCAAACGTCAGGGT-3’ (reverse). The L171C
mutation was introduced by PCR using two primers 5’-GGTGATTGCAGCACCGCGAAAGTT
GAA-3’ (forward) and 5’-GGTGCTGCAATCACCGATCACTTCGAA-3’ (reverse).
An expression vector for PCNA3WT, pET15b+PCNA3WT, was constructed as described
for the construction of pET15b+PCNA1WT, except that two primers 5’-GGAATTCATATGATCT
ACCTGAAATCTTTCGAACG-3’ (forward) and 5’-CGGGATCCGCTCTTCACACTTTCGGC
GCCAGCAG-3’ (reverse) were used to amplify the PCNA3 encoding gene. The R112C mutation
was introduced by PCR using two primers 5’-AACGTTTGCAATCTGGAAGTGTCTGAA-3’
(forward) and 5’-CAGATTGCAAACGTTAAATTCACGAT T-3’ (reverse). The T180C mutation
was introduced by PCR using two primers 5’-AAAGATTGCGGCGGTCTGCAGGATCTG-3’
2
(forward) and 5’-ACCGCCGCAATCTTTAGAAAATTCCAC-3’ (reverse).
We modified pET-15b(+) and pASK-IBA3 plus in advance to constructed an expression
plasmid for the PCNA3-P450cam fusion protein. pET-15b(+) was linearized by PCR using two
primers
5’-ATTCGAACGCCAGCACATGGACAGCGGCAGCAGCGGCCTGGTGC-3’
(forward) and 5’-AGCAGCGGTTTCTTTGCCCATGGTATATCTCCTTCTTAAAGTTAAAC-3’
(reverse). The linearized plasmid was phosphorylated by T7 polynucleotide kinase and
self-ligated by T4 DNA ligase. The resulting plasmid, pStag, has an N-terminal S•Tag sequence.
pASK-IBA3 plus was linearized by PCR using two primers 5’-ATGGGAATTCAGCGCTTGGA
GCCACC-3’ (forward) and 5’-GCCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTT
C-3’ (reverse) to introduce the NcoI and EcoRI sites. The linearized plasmid was phosphorylated
by T7 polynucleotide kinase and self-ligated by T4 DNA ligase. The resulting plasmid was
linearized again by PCR using two primers 5’-GGATCCTGTTTAAACGACCTGTGAAGTGAA
AAATGGCG-3’ (forward) and 5’-GCTTATTCGAACTGCGGGTGGCTCCAAG-3’ (reverse)
and fused with t0 terminator, which was amplified from pQE80L-Kan by PCR using two primers
5’-GCAGTTCGAATAAGCTTAATTAGCTGAGCTTGGACTC-3’ (forward) and 5’-GTTTAAA
CAGGATCCGGATTCTCACCAATAAAAAACGCCC-3’ (reverse), by In-Fusion enzyme. The
resulting plasmid, pASKt, was linearized by PCR using two primers 5’-GCCTTTTTACGGTTC
CTGGC-3’ (forward) and 5’-TTTCTACGGGGTCTGACGC-3’ (reverse) and fused with a p15A
replicon
sequence,
which
was
amplified
from
pLysS
using
two
primers
5’-
CAGACCCCGTAGAAAAGCGCTAGCGGAGTGTATAC-3’ (forward) and 5’-GAACCGTAAA
AAGGCAGAATTACAACTTATATCGTATGGGG-3’ (reverse), by In-Fusion enzyme. The
resulting plasmid, pASKt15, has a p15A replicon.
3
The gene encoding P450cam was amplified by PCR using two primers 5’-GGAATTCG
CTCTTCAGTTGGCGGTAGCATGACGACTGAAACCATACAAAGCAAC-3’ (forward) and
5’-CGGGATCCTTATACCGCTTTGGTAGTCGCC-3’ (reverse). The generated DNA fragment
was cloned into pHSG398 between the EcoRI and BamHI sites. The PmeI site was inserted after
the stop codon by PCR using two primers 5’-TAAGTTTAAACGGATCCTCTAGAGTCGAC-3’
(forward) and 5’-ATCCGTTTAAACTTATACCGCTTTGGTAGT-3’ (reverse) to obtain plasmid
pHSG+GS-P450cam. The gene encoding PCNA3, which was amplified by PCR using two
primers 5’-GGAATTCATATGATCTACCTGAAATCTTTCGAACG-3’ (forward) and 5’-CGGG
ATCCGCTCTTCACACTTTCGGCGCCAGCAG-3’
pHSG+GS-P450cam
between
the
EcoRI
and
(reverse),
SapI
sites.
was
The
cloned
resulting
into
plasmid,
pHSG+PCNA3-P450cam, was digested with NdeI amd BamHI, and the generated fragment was
cloned into pStag that was digested with the same restriction enzymes. The resulting plasmid,
pStag+P3C, was digested with NcoI and PmeI, and the generated fragment was cloned into
pASKt15 that was digested with the same restriction enzymes. The resulting plasmid,
pASKt15+P3C, expresses PCNA3WT-P450cam. The R112C and T180C mutations were
introduced as described above.
The gene encoding PCNA1 was amplified by PCR using two primers 5’-CGGGATCCA
TATGTTTAAAATTGTGTATCCGAACGCC-3’ (forward) and 5’-GGAATTCGCTCTTCACAG
GCGCGGCGCAATCC-3’ (reverse). The generated DNA fragment was cloned into pHSG398
between the BamHI and EcoRI sites to generate the plasmid pHSG+PCNA1. The gene encoding
PdR, which was amplified from pT1R[S1] by PCR using two primers 5’-CGGGATCCGCTCTTC
GCTGGGTGGCGGCGGTAGC-3’ (forward) and 5’-GGAATTCTCAGGCACTACTCAGTTCA
4
GC-3’ (reverse), was digested with EcoRI and SapI, and cloned into pHSG+PCNA1, which was
digested with the same restriction enzymes. The resulting plasmid, pHSG+PCNA1-PdR, was
digested with NdeI and BamHI. The generated DNA fragment was cloned into pET-28b(+)
between the NdeI and BamHI sites. The resulting plasmid, pET28b+P1R, was digested with NcoI
and EcoRI, and the generated fragment was cloned into pASKt that was digested with the same
restriction enzymes. After the resulting plasmid, pASKt+P1R, was digested with NcoI and PmeI,
the generated fragment was ligated into pBAD202/D/lacZ that was digested with the same
restriction enzymes. The resulting plasmid, pBAD+P1R, expresses PCNA1WT-PdR. The G106C
mutation was introduced as described above.
The gene encoding the C73S/C85S mutant of PdX was amplified from pEX[S2] by PCR
using two primers 5’-GGAATTCCTGGTGCCGCGCGGCAGCGGCGGTGGTGGCTCTATGT
CTAAAGTAGTGTATGTGTCAC-3’
(forward)
and
5’-GCTAGTTATTGCTCAGCGG-3’
(reverse). The generated fragment was further amplified by PCR using two primers 5’-GGAATT
CGCTCTTCAGACGGCGGTGGCGGTAGCCTGGTGCCGCGCGGC-3’ (forward) and 5’-GCT
AGTTATTGCTCAGCGG-3’ (reverse). The generated fragment was cloned into pHSG398
between the EcoRI and BamHI sites. The PmeI site was inserted after the stop codon by PCR
using two primers 5’-TAAGTTTAAACGGATCCTCTAGAGTCGAC-3’ (forward) and 5’-ATCC
GTTTAAACTTACCATTGCCTATCGGG-3’ (reverse) to obtain the plasmid pHSG+GStGS-PdX.
The gene encoding PCNA2, which was amplified by PCR using two primers 5’-GGAATTCATA
TGATGAAAGCCAAAGTGATCGATG-3’ (forward) and 5’-CGGGATCCGCTCTTCAGTCCG
CGCGCGGTGC-3’ (reverse), was cloned into pHSG+GStGS-PdX between the EcoRI and SapI
sites. The resulting plasmid, pHSG+PCNA2-PdX, was digested with NdeI and BamHI. The
5
generated fragment was cloned into pET-15b(+) that was digested with the same restriction
enzymes. The resulting plasmid, pET15b+P2X, expresses PCNA2WT-PdX. The L171C mutation
was introduced as described above.
Protein Expression and Purification. E. coli T7 Express Iq was transformed with
pET15b+PCNA1WT. A single colony of cells was inoculated into 5 mL LB medium containing
100 mg/L ampicillin and the cells were grown at 37°C until the OD at 600 nm (OD600) reached
1.0. The culture was added to 250 mL TB medium containing 100 mg/L ampicillin and the cells
were grown at 37°C. After the OD600 reached about 0.5, 0.25 mmol of IPTG was added and the
culture was continued at 27°C overnight. The cells were harvested by centrifugation at 6,000×g
for 20 min and resuspended in 20 mM potassium phosphate buffer, pH 7.4, containing 150 mM
KCl, 10 mM imidazole and 1 mM DTT. After the cells were disrupted by ultrasonication, cell
debris was removed by centrifugation at 22,000×g for 30 min. The resulting cell lysate was
loaded on a HisTrap FF crude column. After washing the column with the same buffer,
PCNA1WT was eluted with a linear gradient of imidazole (10-300 mM). The eluted protein was
diluted 7 times with 20 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 5
mM DTT. The diluted protein was loaded on a HiTrap Q FF column. After washing the column
with the above buffer, the protein was eluted with a linear gradient of KCl (0-500 mM). The
eluted protein was concentrated with an Amicon Ultra-15 Centrifugal Unit (10,000 NMWL). The
concentrated protein was subjected to size-exclusion chromatography on a HiLoad 16/600
Superdex 75 pg column with 50 mM potassium phosphate buffer, pH7.4, containing 150 mM
KCl, 1 mM EDTA and 10 mM DTT. The purified PCNA1WT was concentrated with the above
6
centrifugal unit. PCNA1G108C, PCNA1P186C and PCNA1G108C/P186C were expressed and purified as
described above. PCNA2WT, PCNA2E105C, PCNA2L171C, PCNA2E105C/L171C, PCNA3WT,
PCNA3R112C, PCNA3T180C and PCNA3R112C/T180C were expressed and purified as described for
PCNA1WT, except that the eluted protein from the HisTrap column was loaded on a HiTrap Q FF
column without dilution and eluted with a linear gradient of KCl (150-500 mM). The purification
yields of PCNA1WT, PCNA1G108C, PCNA1P186C, PCNA1G108C/P186C, PCNA2WT,
PCNA2E105C,
PCNA2L171C,
PCNA2E105C/L171C,
PCNA3WT,
PCNA3R112C,
PCNA3T180C and PCNA3R112C/T180C were 9.5, 6.3, 5.9, 15, 43, 47, 43, 13, 11, 7.2, 8.1 and
10 mg, respectively.
E. coli T7 Express Iq was transformed with pBAD+P1R. A single colony of cells was
inoculated into 5 mL LB medium containing 50 mg/L kanamycin and the cells were grown at
37°C until the OD600 reached 1.0. The culture was added to 1 L TB medium containing 50 mg/L
kanamycin and the cells were grown at 37°C. After the OD600 reached about 0.5, 200 mg of
arabinose was added and the culture was continued at 27°C overnight. The cells were harvested
by centrifugation at 6,000×g for 20 min and resuspended in 20 mM potassium phosphate buffer,
pH 7.4, containing 150 mM KCl, 10 mM imidazole and 1 mM DTT. After the cells were
disrupted by ultrasonication, cell debris was removed by centrifugation at 22,000×g for 30 min.
The resulting cell lysate was loaded on a HisTrap FF crude column. After washing the column
with the same buffer, PCNA1WT-PdR was eluted with a linear gradient of imidazole (10-300
mM). The eluted protein was diluted 3 times with 20 mM potassium phosphate buffer, pH 7.4,
containing and 5 mM DTT. The diluted protein was loaded on a HiTrap Q FF column. After
washing the column with 20 mM potassium phosphate buffer, pH 7.4, containing 50 mM KCl
7
and 5 mM DTT, the protein was eluted with a linear gradient of KCl (50-500 mM). The fractions
with a ratio of A280/A455 less than 10 were combined and concentrated with an Amicon Ultra-15
Centrifugal Unit (50,000 NMWL). The concentrated protein was subjected to size-exclusion
chromatography on a HiLoad 16/600 Superdex 200 pg column with 50 mM potassium phosphate
buffer, pH7.4, containing 150 mM KCl and 10 mM DTT. The fractions with a ratio of A280/A455
less than 7.9 were combined and concentrated with the above centrifugal unit. PCNA1G108C-PdR
was expressed and purified as described above. The concentrations of PCNA1WT-PdR and
PCNA1G108C-PdR were calculated using 455 = 11.0 mM-1 cm-1.[S1]
PCNA2WT-PdX and PCNA2L171C-PdX were expressed and purified as described for
PCNA2WT, except that cells were cultured in 1 L TB medium, protein expression was induced by
1 mmol of IPTG, buffers did not contain EDTA, the fractions with a ratio of A412/A280 higher than
0.25 were collected after the anion exchange chromatography, size-exclusion chromatography
was performed on a HiLoad 16/600 Superdex 200 pg column and the fractions with a ratio of
A412/A280 higher than 0.30 were collected after the size-exclusion chromatography. The
concentrations of PCNA2WT-PdX and PCNA2L171C-PdX were calculated using 412 = 11.0 mM-1
cm-1.[S3]
E. coli T7 Express Iq was transformed with pASKt15+P3C. A single colony of cells was
inoculated into 5 mL LB medium containing 100 mg/L ampicillin and the cells were grown at
37°C until the OD600 reached 1.0. The culture was added to 1 L TB medium containing 100 mg/L
ampicillin and the cells were grown at 37°C. After the OD600 reached about 0.5, 200 g of
anhydrotetracycline and 1 mmol of ALA were added and the culture was continued at 27°C
overnight. The cells were harvested by centrifugation at 6,000×g for 20 min and resuspended in
8
20 mM potassium phosphate buffer, pH 7.4, containing 5 mM DTT and 5 mM d-camphor. After
the cells were disrupted by ultrasonication, cell debris was removed by centrifugation at
22,000×g for 30 min. The supernatant was subjected to ammonium sulfate fractionation.
PCNA3WT-P450cam precipitated at 30% saturation. The precipitated protein was dissolved in
20 mM potassium phosphate buffer, pH 7.4, containing 5 mM DTT and 5 mM d-camphor and
dialyzed against the same buffer. The dialyzed protein was loaded on a DE52 column (2.5×8.5
cm). After washing the column with 20 mM potassium phosphate buffer, pH 7.4, containing 150
mM KCl, 5 mM DTT and 5 mM d-camphor, the protein was eluted with a linear gradient of KCl
(150-500 mM). The red fractions were combined and diluted 2 times with 20 mM potassium
phosphate buffer, pH 7.4, containing 5 mM DTT and 5 mM d-camphor. The diluted protein was
loaded on a HiTrap Q FF column. After washing the column with 20 mM potassium phosphate
buffer, pH 7.4, containing 150 mM KCl, 5 mM DTT and 5 mM d-camphor, the protein was
eluted with a linear gradient of KCl (150-500 mM KCl). The fractions with a ratio of A392/A280
higher than 0.75 were combined and diluted 2 times with 50 mM potassium phosphate buffer, pH
7.4, containing 5 mM DTT and 5 mM d-camphor. After washing the column with the above
buffer, the protein was eluted with a linear gradient of potassium phosphate (50-500 mM). The
fractions with a ratio of A392/A280 higher than 1.0 were combined and concentrated with an
Amicon Ultra-15 Centrifugal Unit (50,000 NMWL). The concentrated protein was subjected to
size-exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column with 50 mM
potassium phosphate buffer, pH7.4, containing 150 mM KCl, 10 mM DTT and 5 mM d-camphor.
The fractions with a ratio of A392/A280 higher than 1.1 were combined and concentrated with the
above
centrifugal
unit.
The
concentrations
9
of
PCNA3WT-P450cam
and
PCNA3R112C/T180C-P450cam were calculated using 392 = 90.2 mM-1 cm-1.[S1]
10
Protein Sequences
PCNA1WT-PdR
MGSSHHHHHHSSGLVPRGSHMFKIVYPNAKDFFSFINSITNVTDSIILNFTEDGIFSRHLTEDKV
LMAIMRIPKDVLSEYSIDSPTSVKLDVSSVKKILSKASSKKATIELTETDSGLKIIIRDEKSGAK
STIYIKAEKGQVEQLTEPKVNLAVNFTTDESVLNVIAADVTLVGEEMRISTEEDKIKIEAGEEGK
RYVAFLMKDKPLKELSIDTSASSSYSAEMFKDAVKGLRGFSAPTMVSFGENLPMKIDVEAVSGGH
MIFWIAPRLGGGGSGGGGSMNANDNVVIVGTGLAGVEVAFGLRASGWEGNIRLVGDATVIPHHLP
PLSKAYLAGKATAESLYLRTPDAYAAQNIQLLGGTQVTAINRDRQQVILSDGRALDYDRLVLATG
GRPRPLPVASGAVGKANNFRYLRTLEDAECIRRQLIADNRLVVIGGGYIGLEVAATAIKANMHVT
LLDTAARVLERVTAPPVSAFYEHLHREAGVDIRTGTQVCGFEMSTDQQKVTAVLCEDGTRLPADL
VIAGIGLIPNCELASAAGLQVDNGIVINEHMQTSDPLIMAVGDCARFHSQLYDRWVRIESVPNAL
EQARKIAAILCGKVPRDEAAPWFWSDQYEIGLKMVGLSEGYDRIIVRGSLAQPDFSVFYLQGDRV
LAVDTVNRPVEFNQSKQIITDRLPVEPNLLGDESVPLKEIIAAAKAELSSA
PCNA2WT-PdX
MGSSHHHHHHSSGLVPRGSHMMKAKVIDAVSFSYILRTVGDFLSEANFIVTKEGIRVSGIDPSRV
VFLDIFLPSSYFEGFEVSQEKEIIGFKLEDVNDILKRVLKDDTLILSSNESKLTLTFDGEFTRSF
ELPLIQVESTQPPSVNLEFPFKAQLLTITFADIIDELSDLGEVLNIHSKENKLYFEVIGDLSTAK
VELSTDNGTLLEASGADVSSSYGMEYVANTTKMRRASDSMELYFGSQIPLKLRFKLPQEGYGDFY
IAPRADGGGGSLVPRGSGGGGSMSKVVYVSHDGTRRELDVADGVSLMQAAVSNGIYDIVGDCGGS
ASCATCHVYVNEAFTDKVPAANEREIGMLESVTAELKPNSRLSCQIIMTPELDGIVVDVPDRQW
PCNA3WT-P450cam
MGKETAAAKFERQHMDSGSSGLVPRGSHMIYLKSFERNIRLINMKVVYDDVRVLKDIIQALARLV
DEAVLKFKQDSVELVALDRAHISLISVNLPREMFKEYDVNDEFKFGFNTQYLMKILKVAKRKEAI
EIASESPDSVIINIIGSTNREFNVRNLEVSEQEIPEINLQFDISATISSDGFKSAISEVSTVTDN
VVVEGHEDRILIKAEGESEVEVEFSKDTGGLQDLEFSKESKNSYSAEYLDDVLSLTKLSDYVKIS
FGNQKPLQLFFNMEGGGKVTYLLAPKVGGSMTTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNL
SAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAYEDYRHFSSECPFIPREAGEAYDFI
PTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDYAEPFPIRIFM
LLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVN
GRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIERPERIPAACEELLRRFSL
VADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLG
QHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKA
11
Table
Table S1. Distances between alpha-carbons in disulfide bonds listed in the previous report.[S4]
PDB
ID
1A8E
1ABA
1AHO
1B3A
1BEB
1BQC
1C2A
1CNV
1D0D
Cysteines
Distance (Å)
9C-48C
19C-39C
118C-194C
137C-331C
158C-174C
161C-179C
171C-177C
227C-241C
14C-17C
12C-63C
22C-46C
26C-48C
10C-34C
11C-50C
66C-160C
106C-119C
74C-81C
9C-63C
10C-25C
15C-23C
32C-39C
36C-51C
68C-122C
69C-84C
74C-82C
91C-98C
95C-110C
23C-72C
41C-93C
54C-62C
5C-55C
14C-38C
30C-51C
5.8
6.1
6.0
6.0
5.2
4.6
5.1
4.6
5.2
4.9
5.3
5.6
5.6
6.0
6.1
3.8
5.7
5.2
6.1
4.1
6.3
6.0
5.0
6.1
4.0
6.3
6.2
5.6
5.2
4.9
5.8
5.7
6.4
PDB
ID
1DY5
1E5P
1EDM
1EN2
1EZM
1H03
1HX0
1HXN
1I0V
12
Cysteines
Distance (Å)
26C-84C
40C-95C
58C-110C
65C-72C
38C-42C
57C-149C
51C-62C
56C-71C
73C-82C
3C-18C
12C-24C
17C-31C
35C-39C
49C-64C
58C-70C
63C-75C
82C-86C
30C-58C
270C-297C
7C-48C
34C-64C
69C-111C
97C-127C
28C-86C
70C-115C
141C-160C
378C-384C
450C-462C
229C-432C
338C-380C
390C-407C
2C-10C
6C-103C
5.6
5.5
5.9
5.3
4.6
5.7
5.8
5.6
5.4
6.0
5.8
5.2
5.9
6.0
5.7
5.3
6.0
5.7
4.8
6.0
5.9
5.9
5.5
5.3
5.6
5.1
6.2
5.3
5.7
4.9
6.2
5.3
4.5
PDB
ID
1I4U
1I71
1I8E
1JND
1JU2
1K07
1K5C
1K5N
1ME4
1MSO
1QDD
1QFO
1QNR
Cysteines
Distance (Å)
12C-121C
57C-173C
117C-150C
1C-78C
22C-61C
50C-73C
5C-17C
12C-30C
24C-41C
6C-33C
322C-405C
399C-450C
256C-290C
3C-17C
175C-191C
300C-303C
25C-80C
101C-164C
203C-259C
22C-63C
56C-95C
153C-200C
6C-11C
7C-7C*
19C-20C*
14C-25C
42C-140C
115C-132C
22C-79C
26C-29C
172C-175C
265C-272C
284C-334C
6.1
5.6
5.4
4.9
5.7
6.3
5.8
5.8
5.2
5.9
5.8
6.4
6.4
5.9
5.8
5.9
6.3
6.3
6.4
5.4
4.9
5.4
4.9
4.6
5.3
5.9
5.5
5.1
4.8
5.6
5.4
5.6
6.0
PDB
ID
1QQ4
1SGP
1TML
2DNJ
2MCM
2MSB
2PSP
2TGI
3LZT
3SEB
3SIL
*Intersubunit disulfide bond
13
Cysteines
Distance (Å)
17C-37C
101C-111C
137C-170C
8C-38C
16C-35C
24C-56C
42C-58C
191C-220C
80C-125C
232C-267C
173C-209C
36C-46C
88C-93C
128C-217C
195C-209C
6C-104C
8C-35C
19C-34C
29C-44C
58C-84C
68C-83C
78C-95C
7C-16C
15C-78C
44C-109C
48C-111C
6C-127C
30C-115C
64C-80C
76C-94C
93C-113C
42C-103C
6.3
3.9
4.9
5.2
5.7
4.7
6.5
5.1
5.7
6.1
5.8
3.9
5.6
5.4
5.0
4.9
5.7
6.6
5.9
5.7
6.5
5.9
5.7
5.3
5.8
5.9
5.0
6.1
4.9
5.3
5.5
6.4
Figures
Figure S1. Histogram of distance between alpha-carbons in disulfide bond.
Figure S2. Elution profiles of the mixtures of i) PCNA1WT, PCNA2WT and PCNA3WT (black), ii)
PCNA1P186C, PCNA2E105C and PCNA3WT (red), iii) PCNA1WT, PCNA2L171C and PCNA3R112C (blue),
and iv) PCNA1G108C, PCNA2WT and PCNA3T180C (green) from a HiLoad 16/600 Superdex 200 pg
size exclusion column in 50 mM potassium phosphate buffer, pH 7.4, containing 150 mM KCl, 1
mM EDTA and 10 mM DTT. PCNA3 proteins contained in the mixtures were 1.2 times more than
PCNA1 and PCNA2.
14
Figure S3. Elution profiles of the glutathione-treated heterotrimers composed of i) PCNA1WT,
PCNA2WT and PCNA3WT (black), ii) PCNA1P186C, PCNA2E105C and PCNA3WT (red), iii) PCNA1WT,
PCNA2L171C and PCNA3R112C (blue), and iv) PCNA1G108C, PCNA2WT and PCNA3T180C (green)
from a Superdex 200 10/300 GL size exclusion column in 50 mM potassium phosphate buffer, pH
7.4, containing 150 mM KCl.
Figure S4. Elution profiles of the mixtures of i) PCNA1WT, PCNA2WT and PCNA3WT (black), ii)
PCNA1P186C, PCNA2E105C/L171C and PCNA3R112C (red), iii) PCNA1G108C, PCNA2L171C and
PCNA3R112C/T180C (blue), and iv) PCNA1G108C/P186C, PCNA2E105C and PCNA3T180C (green) from a
HiLoad 16/600 Superdex 200 pg size exclusion column in 50 mM potassium phosphate buffer, pH
7.4, containing 150 mM KCl, 1 mM EDTA and 10 mM DTT. PCNA prtoeins3s contained in the
mixtures were 1.2 times more than PCNA1 and PCNA2.
15
Figure S5. Elution profiles of the glutathione-treated heterotrimers composed of i) PCNA1WT,
PCNA2WT and PCNA3WT (black), ii) PCNA1P186C, PCNA2E105C/L171C and PCNA3R112C (red), iii)
PCNA1G108C, PCNA2L171C and PCNA3R112C/T180C (blue), and iv) PCNA1G108C/P186C, PCNA2E105C and
PCNA3T180C (green) from a Superdex 200 10/300 GL size exclusion column in 50 mM potassium
phosphate buffer, pH 7.4, containing 150 mM KCl.
a)
b)
Figure S6. UV-vis specta of a) PCNA1G108C-PdR (solid line), PCNA2L171C-PdX (dashed line) and
PCNA3R112C/T180C-P450cam (dotted line), and b) PCNA1WT-PdR (solid line), PCNA2WT-PdX
(dashed line) and PCNA3WT-P450cam (dotted line).
16
Figure S7. UV-vis specta of dsPUPPET (solid line) and nPUPPET (dashed line).
Supporting References
[S1] H. Hirakawa, T. Nagamune, ChemBioChem 2010, 11, 1517-1520.
[S2] H. Hirakawa, N. Kamiya, T. Nagamune, Protein Eng. Des. Sel. 2007, 20, 453-459.
[S3] I.F. Sevrioukova, C. Garcia, H. Li, B. Bhaskar, T.L. Poulos, J. Mol. Biol. 2003, 333, 377-392.
[S4] R. Bhattacharyya, D. Pal, P. Chakrabarti, Protein Eng. Des. Sel. 2004, 17, 795-808.
17