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Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris.


ABSTRACT: Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the ?-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by???50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

SUBMITTER: Ali R 

PROVIDER: S-EPMC6875547 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Improved strategy for isoleucine <sup>1</sup>H/<sup>13</sup>C methyl labeling in Pichia pastoris.

Ali Rustam R   Clark Lindsay D LD   Zahm Jacob A JA   Lemoff Andrew A   Ramesh Karthik K   Rosenbaum Daniel M DM   Rosen Michael K MK  

Journal of biomolecular NMR 20190920 12


Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein <sup>1</sup>H/<sup>13</sup>C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pasto  ...[more]

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