UWM Chemistry NMR facility
bar.gif

UWM Logo

Lobster Metallothionein 113Cd3-b Domain Solvent Atom Exposure Patterns Studied by NMR Spectroscopy:
Impact on Metal-Thiolate Cluster Reactivity

bar.gif
bar.gif

Assignment and Structure Determination

Like the holo protein the isolated synthetic Cd3b domains exhibit 113Cd chemical shifts resembling cadmium ions in a tetrahedral environment, coordinated by terminal and bridging cysteines.
Assignment of most proton resonances was obtained using 2D TOCSY and NOESY experiments. The Cd-cysteine connectivities of the Cd3S9 cluster were obtained from heteronuclear 2D-1H{113Cd}HSQC-TOCSY experiments taking advantage of the larger dispersion the HN chemical shifts (Figure 2). The results indicate that the synthetic Cd3b N and Cd3bC exhibit the same metal-coordination as the native holo protein. bar.gif
Figure 2: The HN region of the 2D1H{113Cd} HSQC-TOCSY spectrum (T=298K) shows correlations due to Sg-Cd bonds involving the respective Cys residue and allows one to establish Cd-S cluster connectivities. The advantage to the regular 1H{113Cd}-HSQC or -HMQC experiment is the increased chemical shift dispersion of the amide protons. The delay for INEPT transfer was 8 ms, and a MLEV-16 spinlock of 80 ms was used for TOCSY transfer.
bar.gif A total of 254 and 228 NOE's were obtained for Cd3bN and Cd3b C, respectively from 2D NOESY spectra taken with varying mixing times. Measurement of the coupling constants 3JHNHa, 3JHa Hb and 3JHbCd yielded 13 (11) additional dihedral angle restraints restraints for the backbone angle f, 10 (7) for the side chain dihedral angle c1 and 6 (2) for the CaCbSgCd dihedral angle c2 for Cd3bN (Cd3b C). In addition, 33 restraints for the Cd-S cluster derived from the 1H{113Cd} HSQC-TOCSY experiment were included. Structure calculations were carried out with the program XPLOR employing an ab initio simulated annealing protocol. A subset of 300 structures was generated, and the 30 structures of lowest energy were selected for the final analysis. In the case of Cd3bN a final set of 63 structures exhibited no NOE violation < 0.4D and a superposition yielded a RMSD of 0.36 for the non terminal backbone (residue 4-27), metal cluster and cysteine-C b atoms (1.30 A for all heavy atoms). For Cd3bC 53 structures of lowest energy did not exhibit NOE violations < 0.3 A and RMSD values of 0.38 (1.15) were obtained (Figure 3). bar.gif
Figure3: Superposition of the 30 structures of lowest energy resulting from simulated annealing for Cd3bN and Cd3bC. This figure was prepared using the program MOLMOL2K.1 by Koradi et al [4]
bar.gif
bar.gif
To Top of This Page NMR Home back to NMR lab on line posters To Poster-On-Line
Converted to HTML by F. Holger Försterling March 2001