Amino acid selective cross-saturation method for identification of proximal residue pairs in a protein-protein complex

Shunsuke Igarashi, Masanori Osawa, Koh Takeuchi, Shin Ichiro Ozawa, Ichio Shimada

研究成果: Article査読

12 被引用数 (Scopus)

抄録

We describe an NMR-based approach, the amino acid selective cross-saturation (ASCS) method, to identify the pairs of the interface residues of protein-protein complexes. ASCS uses a "cross-saturation (CS)-donor" protein, in which only one amino acid is selectively 1H-labeled in a 2H-background, and a "CS- acceptor" protein with uniform 2H,15N labeling. Irradiation of the 1H-labeled amino acid, which exists only in the donor, decreases the intensity of the 1H-15N HSQC signals of the acceptor residues proximal to the 1H-labeled CS-source residue(s) through the CS phenomenon. Given the three-dimensional structure of each protein in the complex, but not the complex structure, the combinatorial analysis of multiple ASCS results specify the CS-source residue(s), based on the spatial complementarity between the CS-source residues on the CS donor and the cross-saturated amide protons on the acceptor. NMR investigations of the labeling selectivity and efficiency in an E. coli host, which are critical for ASCS, revealed that Ala, Arg, His, Ne, Leu, Lys, Met, Phe, Pro, Trp, and Tyrare selectively labeled with a high 1H/2H ratio. The observation of the ASCS was then confirmed using the known structure of the yeast ubiquitin (Ub) and yeast ubiquitin hydrolase 1 (YUH1). Conversely, reasonable candidates for the CS-source residues were suggested by the analysis of the ASCS results, with reference to the individual structures of YUH1 and Ub. The pairwise distance information between the CS-source residues and the cross-saturated amide groups obtained by ASCS will be useful for modeling protein-protein complexes.

本文言語English
ページ(範囲)12168-12176
ページ数9
ジャーナルJournal of the American Chemical Society
130
36
DOI
出版ステータスPublished - 2008 9 10
外部発表はい

ASJC Scopus subject areas

  • 触媒
  • 化学 (全般)
  • 生化学
  • コロイド化学および表面化学

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