Determination of the solution three dimensional structure of chemically-synthesized native 113Cd3S9 domain of Lobster Metallothionein by NMR Spectroscopy
Amalia Muñoz*, F. Holger Forsterling, C. Frank Shaw III+ and David H. Petering
Restraints for the chi-1 angles were obtained for those ß-methylen protons for which stereo specific assignments could be made by analysis of the 3Jab coupling constants and the intraresidual HN-Hb and Ha-Hb NOEs.
Figure 4. Alpha/beta proton region of the DQF COSY spectrum of 3 mM Cd3ßC in 5 mM d11-Tris-HCl at pH 7.5.
A Karplus type relation reported12 for the cysteine cluster chi-2 (CaCbSgCd) torsional angles allowed us to obtain additional 5 to 6 non bridging cysteine residues by measuring the 3JHbCd from a couple DQF COSY spectra. Metal cluster connectivities were determined by the 1H{113Cd} HSQC-TOCSY experiment and introduced as a restraints by defining appropiately cluster residues in Xplor. By the first time the cysteine coordination was obtained from the amide region (less populated in signals than the usually used beta-region, alllowing a more easy assignment of cysteine coordination and identification of bridging vs terminal residues in the cluster.
Figure 5.1H{113Cd} HSQC-TOCSY spectrum of 3 mM Cd3ßC in 5 mM d11-Tris-HCl at pH 7.5.
Structure calculations were obtained from the 2D NOESY spectra acquired at 298oK with mixing times of 100, 200 and 300 ms. Assigned NOE were classified according to their peak intensities as follows: 2.8 A (strong), 3.2 A (medium).
Figure 6. Amino acid sequence and summary of the sequential and medium range NOE connectivities.
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Amalia Muñoz - April, 2000