The solution structure refinement of the paramagnetic reduced high-potential iron-sulfur protein I from Ectothiorhodospira halophila by using stable isotope labeling and nuclear relaxation.
The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.
Department of Chemistry, University of Florence, Italy.
I
I
Bertini
M M
MM
Couture
A
A
Donaire
L D
LD
Eltis
I C
IC
Felli
C
C
Luchinat
M
M
Piccioli
A
A
Rosato
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002247:Carbon Isotopes; D002841:Chromatiaceae / Q000737:chemistry* / Q000235:genetics; D006859:Hydrogen / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins; D011487:Protein Conformation; D012996:Solutions; D013816:Thermodynamics
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D007506:Iron-Sulfur Proteins; D009587:Nitrogen Isotopes; D045322:Photosynthetic Reaction Center Complex Proteins; D012996:Solutions; C007943:high potential iron-sulfur protein; D006859:Hydrogen