Complete ¹³C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

Abstract

Complete resonance assignments for the ~sC spectrum of reduced (Cn(I)) rusticyanin have been made using 13C,~SN doubly labeled recombinant material. The reported assignments include those for the earboxyl and carbonyl carbon atoms and protonated aromatic ring carbons, and were obtained using a variety of 2-and 3D inverse-detected NMR experiments, including 13C,ISN,IH triple resonance experiments and HCCH-COSY and -TOCSY. Backbone carbonyl assignments were obtained using 3D HNCO and HCACO spectra, and modified versions of 2D H(CA)CO and HMBC spectra were used to obtain side-chain carboxyl carbon and methionine z-methyl carbon assignments, respectively. A comparison of the 13C~, ~5C° and 13CO chemical shifts with published 'random coil' values confirms the conclusion reached from a consideration of the 3JnN ~ coupling constants and the pattern of sequential NOEs, that the protein consists largely of B-structure.

Multiple Wavelength Anomalous Diffraction (MAD) Crystal Structure of Rusticyanin: a Highly Oxidizing Cupredoxin with Extreme Acid Stability

“…As in other cupredoxins, cis-prolines occur in rusticyanin, at residues 52, 95, and 104. The configuration of these proline residues is confirmed by NMR results (Hunt et al, 1994;Toy-Palmer et al, 1995). The cis-prolines help define the loop conformations that form the environment of the copper.…”

NMR Solution Structure of Cu(I) Rusticyanin fromThiobacillus ferrooxidans: Structural Basis for the Extreme Acid Stability and Redox Potential

“…Rusticyanin labeled with 15 N alone was obtained from T. ferrooxidans (Hunt et al, 1994), and material uniformly labeled with 13 C and 15 N was obtained by overexpression of a recombinant rusticyanin gene in E. coli grown in labeled M9 minimal medium (Casimiro et al, 1995;Toy-Palmer et al, 1995). Samples routinely contained between 2 and 5 mM rusticyanin in 5 mM ascorbic acid and 1 mM H2SO4 (pH 3.4).…”

“…All spectra were Fourier transformed and analyzed using FELIX 2.3 (Biosym Technologies). The 2D and 3D spectra in 90% 1 H2O/10% 2 H2O or in 99% 2 H2O were acquired as described (Hunt et al, 1994;Toy-Palmer et al, 1995); 2D 1 H NOESY was zero-filled to give 2048 × 256 real matrix, while the 3D 15 N and 13 C NOESY-HSQC spectra generally contained 64 planes of 1024 × 1024 real data points. The gradientenhanced 4D 13 C NOESY spectrum (Vuister et al, 1993a) was acquired at 500 MHz.…”

“…Distance constraints for calculation of 3D structures were generated using information from 3D 15 N NOESY-HSQC, 3D and 4D 13 C NOESY-HSQC and 2D 1 H NOESY spectra, and the cross-peaks were assigned according to the published resonance assignments (Hunt et al, 1994;Toy-Palmer et al, 1995) with the aid of the program XPKASGN, written by Xiang Li. An initial distance geometry calculation was made using only unambiguous cross-peaks.…”

“…The global fold of the protein at pH 3 was shown to be that of a compact b-barrel with a hydrophobic core particularly rich in aromatic residues. Complete 1 H, 13 C and 15 N resonance assignments have been made for Cu(I) rusticyanin, with the aid of uniformly 13 C, 15 N-labeled protein obtained from the over-expression in Escherichia coli of a synthetic gene (Casimiro et al, 1995;Toy-Palmer et al, 1995). Here, we report the results of a structure calculation using distance and dihedral angle constraints derived from two, three and four-dimensional NMR spectra of Cu(I) rusticyanin in the distance geometry program DISGEO (Havel & Wü thrich, 1984), followed by restrained molecular dynamics/simulated annealing using AMBER (Pearlman et al, 1995).…”