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Default Protein structure and analysis site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR.

Protein structure and analysis site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR.

Related Articles Protein structure and analysis site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR.

Anal Biochem. 2019 Mar 22;:

Authors: Inaba S, Shiota A, Yoshida T, Oda M

Abstract
One of the reasons it is difficult to analyze protein structural dynamics at atomic resolution using NMR is the molecular size of the protein. The selective amino acid labeling method is one of the effective methods that can solve this problem. In this study, to determine the site-specific conformational change in 3?-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3?HSD), which forms a dimer composed of two 26 kDa subunits, we expressed and purified 15N-Tyr labeled Ps3?HSD and its mutants, and analyzed the conformational change upon NADH binding. Using the Tyr substituted mutants, we first assigned the respective signals of four Tyr residues. In the titration experiments with NADH, the four Tyr signals changed uniquely; changes in chemical shift and signal broadening were observed. The NADH binding affinity, determined from the plots of the 1H and 15N chemical shift changes, was comparable to those reported previously. Together with the crystal structure information for Ps3?HSD in the NADH-free and -bound states, site-specific conformational changes including environmental changes could be deduced.


PMID: 30910701 [PubMed - as supplied by publisher]



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