Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

Lindsay Clark; Jacob A. Zahm; Rustam Ali; Maciej Kukula; Liangqiao Bian; Steven M. Patrie; Kevin H. Gardner; Michael Rosen; Daniel M. Rosenbaum

doi:10.1007/s10858-015-9939-2

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

Lindsay Clark, Jacob A. Zahm, Rustam Ali, Maciej Kukula, Liangqiao Bian, Steven M. Patrie, Kevin H. Gardner, Michael Rosen, Daniel M. Rosenbaum^*

^*Corresponding author for this work

Chemistry

Research output: Contribution to journal › Article › peer-review

41 Scopus citations

Abstract

¹³C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific ¹³C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient ¹³C isoleucine d1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

Original language	English (US)
Pages (from-to)	239-245
Number of pages	7
Journal	Journal of Biomolecular NMR
Volume	62
Issue number	3
DOIs	https://doi.org/10.1007/s10858-015-9939-2
State	Published - Jul 1 2015

Funding

Funding was provided by a National Science Foundation Predoctoral Fellowship (Grant No. 1000136529 to L.C.), the Welch Foundation (I-1770 to D.M.R, I-1544 to M.K.R., I-1424 to K.H.G.), the Searle Scholars Program (D.M.R), a Packard Foundation Fellowship (D.M.R), the National Institutes of Health (T32 GM008297 supporting J.Z., R01 GM106239 to K.H.G., R01-GM56322 to M.K.R) and the Howard Hughes Medical Institute (M.K.R.).

Keywords

Actin
Deuteration
Eukaryotic expression system
Methyl labeling
Pichia pastoris
TROSY

ASJC Scopus subject areas

Biochemistry
Spectroscopy

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abstract = "13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13C isoleucine d1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.",

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AU - Ali, Rustam

AU - Kukula, Maciej

AU - Bian, Liangqiao

AU - Patrie, Steven M.

AU - Gardner, Kevin H.

AU - Rosen, Michael

AU - Rosenbaum, Daniel M.

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PY - 2015/7/1

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Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

Abstract

Funding

Keywords

ASJC Scopus subject areas

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