Signal assignment and secondary structure analysis of a uniformly [¹³C, ¹⁵N]-labeled membrane protein, H⁺-ATP synthase subunit c, by magic-angle spinning solid-state NMR

Authors: Kobayashi, Masatoshi; Matsuki, Yoh; Yumen, Ikuko; Fujiwara, Toshimichi; Akutsu, Hideo

Source: Journal of Biomolecular NMR, Volume 36, Number 4, December 2006 , pp. 279-293(15)

Publisher: Springer

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Abstract:

Signal assignment and secondary structural analysis of uniformly [¹³C, ¹⁵N] labeled H⁺-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the ¹³C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue ¹³C-¹³C and ¹³C-¹⁵N chemical shift correlations, 78% of <EquationSource Format="TEX"><![CDATA[$${rm C}^{upalpha}$$]]></EquationSource> , 72% of <EquationSource Format="TEX"><![CDATA[$${rm C}^{upbeta}$$]]></EquationSource> , 62% of C′ and 61% of N^H signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific assignment. On top of this, the ¹³C-¹³C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by <EquationSource Format="TEX"><![CDATA[$$upalpha$$]]></EquationSource> -helices with a loop structure in the middle (at sequence 41-45) as in the case of the solution structure in spite of additional extended conformations at 76-79 at the C-terminus.

Keywords: transmembrane protein; FO subunit c; MAS solid-state NMR; non-crystal solid; sequential assignment; spectral simulation

Document Type: Research article

DOI: http://dx.doi.org/10.1007/s10858-006-9094-x

Affiliations: 1: Email: akutsu@protein.osaka-u.ac.jp

Publication date: 2006-12-01