Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative [2Fe-2S]ferredoxin.
Two alternative T7 RNA promoter/polymerase systems have been employed for the heterologous expression of a plant-type [2Fe-2S]ferredoxin, Anabaena 7120 vegetative ferredoxin, in Escherichia coli at high levels (approximately 20 mg/liter of culture). One system was used when 15N-labeling the ferredoxin uniformly by growing E. coli with 15NH4Cl as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating 2H-, 13C-, and 15N-labeled amino acids. The labeled ferredoxin samples were studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Results from 1H and 2H NMR studies of samples containing [2H alpha]Cys, [2H beta 2, beta 3]Cys, [13 C beta]-Cys, and [15N]Cys have confirmed previous cysteinyl proton resonance assignments (L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M. Holden, B. L. Jacobson, I. Rayment, and J. L. Markley (1991) Biochemistry 30, 7363-7368). All four 13C NMR peaks arising from the four cysteinyl beta-carbons and all four 15N NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys46, which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of 13Ci-15Ni+1 coupling in a ferredoxin sample with incorporated [13C']Ala and [15N]Cys. The nitrogen signal of Cys 41 was assigned tentatively on the basis of its chemical shift and T1 relaxation time. The cysteinyl beta-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) beta-protons. The beta-carbons resonance from Cys46 in the oxidized state has been assigned by its correlation with the corresponding resonance in the reduced state; this was accomplished by following the progressive air oxidation of a protein sample reduced by dithionite in the presence of methyl viologen. The spin-lattice relaxation times of the beta-carbons of the two cysteines coordinated to Fe)III) were similar in the oxidized and reduced states. This suggests that the antiferromagnetic coupling present in the reduced cluster has little influence on the electronic relaxation time of the Fe(III). Studies of the temperature dependence of the 1H, 13C, and 15N signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (delta delta/delta T-1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although delta delta/delta T-1 is positive for Cys49 1H beta 2 and 1H beta 3, it is negative for Cys49 13C beta. Although delta delta/delta T-1 is negative for protons of cysteines ligated to Fe(II) and positive for protons of cysteines ligated to Fe(III), it is positive for all the cysteinyl nitrogens.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
H
H
Cheng
W M
WM
Westler
B
B
Xia
B H
BH
Oh
J L
JL
Markley
D017033:Anabaena / Q000737:chemistry* / Q000235:genetics; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002247:Carbon Isotopes; D002352:Carrier Proteins; D003545:Cysteine / Q000737:chemistry; D003903:Deuterium; D005288:Ferredoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007553:Isotope Labeling; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D002352:Carrier Proteins; D005288:Ferredoxins; D008024:Ligands; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D001120:Arginine; D003903:Deuterium; D003545:Cysteine