NMR-Based Screening of Proteins Containing 13C-Labeled Methyl Groups

Autor:	and Stephen F. Betz, Renaldo Mendoza, Jamey Mack, Philip J. Hajduk, Stephen W. Fesik, David J. Augeri, Jianguo Yang
Rok vydání:	2000
Předmět:	chemistry.chemical_classification Chemistry Stereochemistry Chemical shift A protein General Chemistry Biochemistry Combinatorial chemistry Catalysis Amino acid Colloid and Surface Chemistry Deuterium Valine Isoleucine Leucine
Zdroj:	Journal of the American Chemical Society. 122:7898-7904
ISSN:	1520-5126 0002-7863
DOI:	10.1021/ja000350l
Popis:	A method is described for NMR-based screening that involves monitoring the 13C/1H chemical shift changes of a protein selectively labeled with 13C at the methyl groups of valine, leucine, and isoleucine (δ1 only). Using this approach, the sensitivity is increased by nearly 3-fold compared with that of NMR-based screening using 1H/15N chemical shifts. A synthetic route is described for the inexpensive production of the labeled amino acid precursors [3,3‘-13C]-α-ketoisovalerate and [3-13C]-α-ketobutyrate, making the cost of protein preparation comparable to that of uniform 15N labeling. In addition to enhancing the NMR-based screening efforts directed against low molecular weight proteins (MW ≤ 30 kDa), the use of the selective methyl labels in combination with deuterium labeling is advantageous for screening high molecular weight protein targets (MW ≥ 100 kDa).
Databáze:	OpenAIRE
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