Supporting file
Differential isotope-labeling for Leu and Val residues in a protein by E.
coli cellular expression using stereo-specifically methyl labeled amino
acids
Journal of Biomolecular NMR
Yohei Miyanoiri1, Mitsuhiro Takeda1, Kosuke Okuma2,3, Akira M. Ono2,3, Tsutomu
Terauchi2,3 and Masatsune Kainosho1,2*
1
Structural Biology Research Center, Graduate School of Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan;
2
Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-ohsawa,
Hachioji, 192-0397, Japan;
3
SAIL Technologies Inc., 1-40 Suehiro-cho 1-chome, Tsurumi-ku, Yokohama,
Kanagawa, 230-0045, Japan.
Correspondence should be addressed to:
Masatsune Kainosho
Structural Biology Research Center, Graduate School of Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan
Tel: +81-(0)52-747-6474; Fax: +81-(0)52-747-6433
E.mail: kainosho@tmu.ac.jp
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Content of Supporting File
Table S1. Incorporation efficiencies of the Leu and Val residues in MSG prepared by E.
coli cellular expression with various amounts of exogenous leucine and valine, and the
effects of acetolactate synthase inhibitors on the incorporation efficiencies and protein
expression levels.
Table S2. Comparison of the numbers of Val and Leu methyls within an upper limit of 7
Å, which likely give observable methyl-methyl NOEs, for various MSG samples
prepared using -ketoisovalerates and stereo-specifically methyl labeled amino acids.
Fig. S1. Detection of “13C-labeled” Val residues in MSG prepared by E. coli cellular
expression in deuterated M9 medium composed of [U-13C]-glucose, [U-2H]- Val, and
[U-2H]-Leu, by 2D 1H-13C HMQC spectroscopy.
S2
Table S1. Incorporation efficiencies of the Leu and Val residues in MSG prepared by E.
coli cellular expression with various amounts of exogenous leucine and valine, and the
effects of acetolactate synthase inhibitors on the incorporation efficiencies and protein
expression levels.
a. Acetolactate synthase inhibitors (Ray, 1984; LaRossa and Schloss, 1984).
b. Amounts of purified MSG samples isolated from 30 ml cultures.
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Table S2. Comparison of the numbers of Val and Leu methyls within an upper limit of 7
Å, which are likely to give observable methyl-methyl NOEs, for various MSG samples
prepared using -ketoisovalerates and stereo-specifically methyl labeled amino acids.
 -keto
-isovalerate a
Number of
NOEs b
436
g 1-Val
+
d 2-Leu
g 2-Val
+
d 1-Leu
g 1-Val
g 2-Val
d 1-Leu
d 2-Leu
118
106
15
8
52
57
a. Using the [3-13C1H3; 3,4,4,4-2H4]--ketoisovalerate.
b. The numbers of the Leu and Val methyls within an upper inter-proton distance limit
of 7Å, estimated by the X-ray structure of MSG (PDB ID: 1D8C) (Howard et al., 2000).
We used the geometric center of three protons as the average position of methyl protons.
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Fig. S1. Detection of “13C-labeled” Val residues in MSG prepared by E. coli cellular
expression in deuterated M9 medium composed of [U-13C]-glucose, [U-2H]- Val, and
[U-2H]-Leu, by 2D 1H-13C HMQC spectroscopy.
(a) High-field methyl TROSY signals observed in the 1H-13C HMQC spectrum of MSG,
prepared in deuterated M9 medium composed of 2,000 mg/L [U-2H] glucose,
supplemented with 120 mg/L of [3-13C1H3; 3,4,4,4-2H4]--ketoisovalerate and 70 mg/L
of [4-13C; 3,3-2H2]--ketobutyrate. This selectively [Ile, Leu, Val]-labeled MSG in a
deuterated background shows well-separated methyl signals, including I12 d1, V24g2,
V118 g1/g2, V24 g2, V348 g1, L85 d2, L25 d2, L142 d2, and L230 d2, in the selected
high-field 1H-NMR chemical shift region. The assignments were made according to the
previous publications (Tugarinov and Kay, 2003; Gans et al., 2010).
(b) Methyl signals observed in the identical region of the 2D 2H decoupled 1H-13C
HMQC spectrum of MSG, prepared in deuterated M9 medium composed of 2,000 mg/L
[U-13C] glucose, supplemented with 100 mg/L [U-2H]Val, and 20 mg/L [U-2H] Leu. By
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referring to the assignments of the Ile d1, Valg1/g2, and Leu d1/d2 methyl signals in panel
(a), and also according to the previous assignments for the Ile g2 and Ala  methyl
signals (Sheppard et al., 2009; Ayala et al. 2012), we were able to identify all of the
signals in this region, as shown in the spectrum. In addition to the Ile d1 and Valg1/g2
methyl signals, which were also observed in panel (a), five Ile g2 methyls and one Ala
methyl were observed and assigned (shown in green letters). A comparison of the signal
intensities for the Val g1/g2 signals to those for Ile g2/d1 and Ala  revealed that the 13C
incorporation efficiencies for the Val methyls were much smaller. Note that all of the Ile
and Ala residues in this MSG preparation should be biosynthetically derived from
[U-13C]- glucose (98%
(>98%
13
13
C) in 2H2O (>99% 2H); therefore, they are highly 13C-labeled
C) and partially deuterated (~85% 2H). Since approximately 80% of the Val
residues were from the exogenous [U-2H]-valine (100 mg/L), less than 20% of them
were from the partially deuterated [U-13C]-valine synthesized from [U-13C]-glucose in
2
H2O (Table S1). It is also plausible that there were no observable 1H-13C signals for the
Leu d2 methyls, since the conversion from partially deuterated [U-13C]-valine into
partially deuterated [U-13C]-leucine is less than 10%, due to the inhibiting effect of
exogenous [U-2H]-leucine (20 mg/L) (Table S1). Therefore, the
13
C-incorporation
efficiency for the Leu residues should be no more than 2%, which is on the order of the
natural abundance level of
13
C (1.1%). We found that the
13
C-chemical shifts for the
methyl signals were ~0.6 ppm higher than those for panel (a), due to the deuterium
isotope shift for directly bonded 13C, showing that all of the observed Ile d1 and Val g1/g2
methyl signals in panel (b) were due to “13C1HD2” isotopomers. This is reasonable,
since the residual proton concentrations for proteins expressed under these conditions
are known to be around 15% on average (Rosen et al., 1996; Shekhtman et al., 2002;
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Otten et al., 2010; Guo and Tugarinov, 2010).
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