Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2007
©
Copyright Wiley
-
VCH Verlag GmbH & Co.
KGaA, 69451 Weinheim, 200
8
Supporting Information
for
Enzym
atic N
-
terminal Addition of Non
canonical Amino Acids to Peptides
and Proteins
R
ebecca
E. Connor, K
onstantin
Piatkov, A
lexander
Varshavsky, and D
avi
d
A. Tirrell*
Methods
Synthesis of
a
zidonorleucine
-
biotin (12
)
:
Azidonorleucine (12.65 mg, 0.073 mmol)
and PFP
-
biotin (30.1 mg, 0.073 mmol) were dissolved in 1.5 mL of a 9:1:5 mixture of
dimethylformamide, dimethylsulfoxide and methanol.
T
riethylamine
(4
equiv
)
was
ad
-
ded and the reaction stirred overnight at room temperature. The reaction was purified
on a C18 Sep
-
Pak column (Waters) using a stepwise gradient from 0.1% trifluoro
-
acetic acid to 100% acetonitrile. The fractions containing the desired produ
ct were
lyophilized to 6.8 mg of a fluffy white powder (23.4% yield). ESI MS (
m/z
): 398.9
[
M
+H]
+
(expected
m/z
: 398.17)
Synthesis of Azido
-
PEG5000
-
Fluorescein (APF, 13)
:
Azidopropylamine was syn
-
thesized as previously described.
1
To prepare the azide
-
funct
ionalized PEG, mSPA
-
PEG
-
fluorescein (29 mg, 6
μ
mol) was dissolved in azidopropylamine (300
μ
L, 2.9
mmol) and allowed to react at room temperature for 2 h. The reaction mixture was
then added dropwise to 20 mL of diethyl ether which caused the PEG reagents
to
form a yellow precipitate. The precipitate was collected by centrifugation and the
ether supernatant decanted. The precipitate was dissolved in 1 mL of methanol and
added dropwise to ether again and the precipitate collected as before.
The reco
-
vered pr
ecipitate was used without further purification. Addition of an azide was con
-
firmed by IR analysis.
Construction of Arg
-
eDHFR
-
HA and G
ly
eDHFR
-
HA plasmids
:
DNA fragments
encoding the gene for
E. coli
dihydrofolate reductase were amplified by PCR from
E.
c
oli
genomic DNA with the following pairs of primers: Oligo 298 (5’
-
AGGCTCCGCG
-
GTGGTcgtAAAATGATCAGTCTGATTGCGGC
–
3’) and Oligo 314 (5’
-
TTTAAGCTT
-
AGGCGTAATCTGGGACATCGTATGGGTAGCCGCTCCCCCGC
CGCTCCAGAATC
T
–
3’) for Arg
-
eDHFR
-
HA; and Oligo 298 and Oligo 301 (5’
-
A
GG
CTC
CGC
GGT
GGT
-
ggtAAAATGATCAGTCTGATTGCGGC
-
3’) for G
ly
eDHFR
-
HA. The resulting frag
-
ments were digested with
Sac
II and
Hind
III, and ligated into
Sac
II
/
Hind
III
-
digested
pHUE
[2]
to generate plasmids pKP141 and pKP144, respectively.
Expression and purification
of recombinant proteins
:
Overnight cultures in
E. coli
strain KPS17
[3]
were subcultured
1:100 into 200 mL Luria broth containing ampicillin
and grown
to a late exponential phase at 37°C. Protein expression
was induced by
adding isopropyl
-
1
-
thio
-
β
-
D
-
galactopyranoside
(IPTG) to a final concentration of 0.1
m
M
. After 3 h, harvested cells were resuspended in 15 mL of buffer
L (50
m
M
Na
2
H
-
PO
4
/NaH
2
PO
4
at pH 7.4, 500
m
M
NaCl, 10
m
M
imidazole), with 1
m
M
phenylmethyl
-
sulf
onyl
fluoride. His
6
-
tagged
recomb
inant proteins were purified by nickel
-
affinity
chromatography
using batch mode under native conditions, based on the QIA
express
protocol (Qiagen). The cells were
lysed by sonication (3
x
1 min bursts at 0°C) and
the
soluble protein fraction recovered by c
entrifugation at 4°C
(15 min at 15
300
g
).
To the supernatant, 2 mL of
a 50% slurry of nickel
-
nitrilotriacetic acid (Ni
-
NTA) aga
-
rose
in buffer L was added; the mixture was then placed on a rotary wheel at 4°C
for
1 h. The lysate/Ni
-
NTA mixture was centrif
uged (5 min at
~550
g
) and the remaining
Ni
-
NTA agarose pellet was washed 5
times in 50 mL buffer W (50
m
M
Na
2
HPO
4
/
NaH
2
PO
4
at pH 7.4, 500
m
M
NaCl, 30
m
M
imidazole). The His
6
-
tagged protein was
eluted
from the Ni
-
NTA resin in 1 mL fractions with buffer L co
ntaining
300
m
M
imid
-
azole. Chosen fractions were pooled and
dialyzed against buffer L, along with deubi
-
quitylating protease Usp2
-
cc
6
for 12 h at 4°C. After cleavage by the protease, nickel
affinity chromatography was used to separate Usp2
-
cc and the His
6
-
tagged ubiquitin
domain from the modified DHFR according to published methods.
[2]
[1]
Carboni, B.; Benalil, A.; Vaultier, M.
J. Org. Chem.
1993
,
58
, 3736
-
3741.
[2]
Catanzariti, A. M.; Soboleva, T. A.; Jans, D. A.; Board, P. G.; Baker,
R. T.
Prot. Sci.
2004
,
13
, 1331
-
9.
[3]
Graciet, E.; Hu, R.G.; Piatkov, K.; Rhee, J.H.; Schwarz, E. M.; Varshavsky, A.
Proc. Natl.
Acad. Sci.
2006
,
103
, 3078
-
3083.
Table S1
.
The percent of modified protein Etf
-
R
-
DHFR after increasing reaction
length as
determined by comparison of the molar amounts of arginine found by Ed
-
man degradation in analyses of the first and second residues. Arginine found as the
first residue arises from unmodified protein, whereas arginine found as the second
residue arises from
modified protein. The reaction is largely complete after 3 h.
Incubation Time [h]
Percent modification
2
80
3
92
4
93
Figure S
1
.
A) Representative reversed
-
phase HPLC analysis (UV detection at 324 nm) of
the transfer of leucine to KA
-
AMC after
incubation. A small amount of the starting material
(a), and the tripeptide prod
uct, LKA
-
AMC, (b) are visible.
B) The ESI mass spectrum of (b)
confirms its identity as LKA
-
AMC, expected
[
M
LKA
-
AMC
+H]
+
=488.28
m/z
and expected
[
M
LKA
-
AMC
+2H]
2+
=244.6
m/z
.
C) MS
/MS analysis of the sing
ly
charged ion of the product further
confirms the N
-
terminal addition of leucine to KA
-
AMC with fragmentation into dipeptide LK,
expected
[
M
LK
]
+
=242.19
m/z
, tripeptide LKA,
[
M
LKA
]
+
=313.22
m/z
, and the aminocoumarin,
[
M
aminocoumarin
+H]
+
=176.06
m/z
.
Figure S
2
. A) Representative reversed
-
phase HPLC analysis of the transfer reaction of tri
-
fluoroleucine
2
(Tfl) to KA
-
AMC using UV detection at 324 nm. The starting material is not
detectable at 17.5 min and the isomeric produc
t (b) ap
pears at 19.6 minutes.
B) Analysis of
peak (b) by ESI mass spectrometry confirms that it contains the tripeptide product, Tfl
-
KA
-
AMC,
expected
[
M
Tfl
-
KA
-
AMC
+H]
+
=542.25
m/z
and
[
M
Tfl
-
KA
-
AMC
+2H]
2+
=271.67
m/z.
C) MS/MS
analysis of the sing
ly
charged ion of t
he product further confirms the N
-
terminal addition of Tfl
to KA
-
AMC with fragmentation into dipeptide Tfl
-
K, expected
[
M
Tfl
-
K
]
+
=296.16
m/z
and the
tripeptide Tfl
-
KA, expected
[
M
Tfl
-
KA
]
+
=367.2
m/z.
Figure S
3
.
A) Representative reversed
-
phase HPLC analys
is of the transfer reaction of de
-
hydroleucine
1
(Ddl) to KA
-
AMC using UV detection at 324 nm. Both the starting material (a)
and the tripe
ptide product (b) are visible.
B) Analysis of peak (b) by ESI mass spectrometry
confirms that it contains the tripept
ide product, Ddl
-
KA
-
AMC,
expecte
[
M
Ddl
-
KA
-
AMC
+H]
+
=
486.26
m/z
and expected
[
M
Ddl
-
KA
-
AMC
+2H]
2+
=243.63
m/z.
C)
MS/MS analysis of the singly
charged ion of the product further confirms the N
-
terminal addition of Ddl to KA
-
AMC with
fragmentation into dipeptid
e Ddl
-
K,
[
M
Ddl
-
K
]
+
=240.17
m/z
and the tripeptide Ddl
-
KA,
[
M
Ddl
-
KA
]
+
=
311.21
m/z.
Figure S
4
.
A) Representative reversed
-
phase HPLC analysis of the transfer reaction of oxo
-
norvaline
3
(Oxo) to the dipeptide. Both the starting material (a) and the tripe
ptide product (b)
are visible. B) The ESI mass spectra of peak (b) confirms that it contains the tripeptide
-
aminocoumarin product, expected
[
M
Zxo
-
KA
-
AMC
+
H]
+
= 488.24
m/z.
C) Fragmentation of the tri
-
peptide
-
AMC product ion results in production of the trip
eptide ion, Oxo
-
KA, expected
[
M
Oxo
-
KA
]
+
=312.19
m/z
, as well as loss of water,
m/z
= 470, and ammonium,
m/z
= 453 from the
parent ion at 488.12
m/z
.