Abstract
A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and (1H-γ methyl)- valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≃100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)- isoleucine (>90% incorporation), 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 369-374 |
| Number of pages | 6 |
| Journal | Journal of Biomolecular NMR |
| Volume | 13 |
| Issue number | 4 |
| DOIs | |
| State | Published - 1999 |
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Keywords
- α-ketobutyrate
- α-ketoisovalerate
- Deuteration
- Methyl protonation
ASJC Scopus subject areas
- Spectroscopy
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
Cite this
A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. / Goto, Natalie K.; Gardner, Kevin H.; Mueller, Geoffrey A.; Willis, Randall C.; Kay, Lewis E.
In: Journal of Biomolecular NMR, Vol. 13, No. 4, 1999, p. 369-374.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins
AU - Goto, Natalie K.
AU - Gardner, Kevin H.
AU - Mueller, Geoffrey A.
AU - Willis, Randall C.
AU - Kay, Lewis E.
PY - 1999
Y1 - 1999
N2 - A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and (1H-γ methyl)- valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≃100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)- isoleucine (>90% incorporation), 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
AB - A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and (1H-γ methyl)- valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≃100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)- isoleucine (>90% incorporation), 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
KW - α-ketobutyrate
KW - α-ketoisovalerate
KW - Deuteration
KW - Methyl protonation
UR - http://www.scopus.com/inward/record.url?scp=0032898682&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032898682&partnerID=8YFLogxK
U2 - 10.1023/A:1008393201236
DO - 10.1023/A:1008393201236
M3 - Article
C2 - 10383198
AN - SCOPUS:0032898682
VL - 13
SP - 369
EP - 374
JO - Journal of Biomolecular NMR
JF - Journal of Biomolecular NMR
SN - 0925-2738
IS - 4
ER -