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Methods Mol Biol. 2017;1495:131-145.

Segmental Isotopic Labeling of Proteins for NMR Study Using Intein Technology.

Author information

1
iHuman Institute, ShanghaiTech University, 99 Haike Road, Pudong, Shanghai, 201203, China.
2
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA. david.cowburn@einstein.yu.edu.

Abstract

Segmental isotopic labeling of samples for NMR studies is attractive for large complex biomacromolecular systems, especially for studies of function-related protein-ligand interactions and protein dynamics (Goto and Kay, Curr Opin Struct Biol 10:585-592, 2000; Rosa et al., Molecules (Basel, Switzerland) 18:440, 2013; Hiroaki, Expert Opin Drug Discovery 8:523-536, 2013). Advantages of segmental isotopic labeling include selective examination of specific segment(s) within a protein by NMR, significantly reducing the spectral complexity for large proteins, and allowing for the application of a variety of solution-based NMR strategies. By utilizing intein techniques (Wood and Camarero, J Biol Chem 289:14512-14519, 2014; Paulus, Annu Rev Biochem 69:447-496, 2000), two related approaches can generally be used in the segmental isotopic labeling of proteins: expressed protein ligation (Muir, Annu Rev Biochem 72:249-289, 2003) and protein trans-splicing (Shah et al., J Am Chem Soc 134:11338-11341, 2012). Here, we describe general implementation and latest improvements of expressed protein ligation method for the production of segmental isotopic labeled NMR samples.

KEYWORDS:

Expressed protein ligation; Intein; Isotopic labeling; NMR; Structural biology

PMID:
27714614
DOI:
10.1007/978-1-4939-6451-2_9
[Indexed for MEDLINE]

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