Open AccessSolution structure of the functional domain of Paracoccus denitrificans cytochrome c 552 in the reduced state
Author(s) -
Pristovšek Primož,
Lücke Christian,
Reincke Britta,
Ludwig Bernd,
Rüterjans Heinz
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01456.x
Subject(s) - heteronuclear molecule , paracoccus denitrificans , two dimensional nuclear magnetic resonance spectroscopy , homonuclear molecule , chemistry , crystallography , stereochemistry , side chain , heme , cytochrome , protein secondary structure , moiety , nuclear magnetic resonance spectroscopy , molecule , biochemistry , organic chemistry , enzyme , polymer
In order to determine the solution structure of Paracoccus denitrificans cytochrome c 552 by NMR, we cloned and isotopically labeled a 10.5‐kDa soluble fragment (100 residues) containing the functional domain of the 18.2‐kDa membrane‐bound protein. Using uniformly 15 N‐enriched samples of cytochrome c 552 in the reduced state, a variety of two‐dimensional and three‐dimensional heteronuclear double‐resonance NMR experiments was employed to achieve complete 1 H and 15 N assignments. A total of 1893 distance restraints was derived from homonuclear 2D‐NOESY and heteronuclear 3D‐NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 ± 0.10 Å and 1.09 ± 0.09 Å for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N‐terminus and C‐terminus and three shorter helices surrounding the heme moiety; the helices are connected by well‐defined loops. Comparison with the X‐ray structure shows some minor differences in the positions of the Trp57 and Phe65 side‐chain rings as well as the heme propionate groups.