Lobster Metallothionein 113Cd3-b Domain Solvent Atom Exposure
and HN-S Hydrogen Bonding
Patterns Studied by NMR Spectroscopy:
Impact on Metal-Thiolate Cluster Reactivity
Amalia Muñoz#, F. Holger Försterling, C. Frank Shaw III+ and David Petering Department of Chemistry and UWM-NIEHS Marine and Freshwater Biomedical Core Center,
University of Wisconsin-Milwaukee, Milwaukee WI 53211
# Current address: Dept. of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706
+ Current address: Dept. of Biochemistry, University of Eastern Kentucky, Richmond KY 40475
Metallothionein (MT) is a cysteine-rich metal-binding protein that binds six or seven d10 metal ions. MT's of
different origins exhibit a range of highly conserved cysteine positions in their sequence and show differences
in metal-cysteine connectivities and reactivity. 113Cd6-Lobster-MT, which includes two
Cd3S9b -domains, was chosen as a basic model to study the structure-function relationship among
b domains. It was previously found that the reactivities toward EDTA and DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid))
differ between the two domains (bC and bN)
with the bC domain the less reactive
one.
The metal-cysteine coordination of the isolated domains were defined from 2D 1H/
113Cd HSQC-TOCSY experiments and found to be the same as in the native holo-Cd6-MT from lobster
and crab. 1H/113Cd HSQC-TOCSY allowed us to easily identify the bridging and terminal
cysteines from the amide proton region. Proton resonances were assigned using two dimensional methods; the
structure calculation was performed using proton-proton distance restraints from NOE data and restraints for the
and 1 dihedral angles derived from 3JHNH and 3JHH In addition
restraints for some Cys-Cd 2 angles derived from heteronuclear 3JHCd couplings were
included. HN-S hydrogen bonds were defined using a long range optimized 2D 1H/113
Cd HMQC experiment and quantified using 1D 1H/113
Cd heteronuclear spin echo difference experiments. The results shown here indicate than the N-domain
has a more labile structure than the C. A detailed analysis of the solvent accessibility of the cluster
sulfur atoms in the two domains revealed differences between the two domains with the bC
domain exhibiting less solvent access to the Cd3S9 cluster than the bN
consistent with the lower reactivity observed. A detailed discussion of the relationship of Cys-sulfur
solvent accessibility, cluster reactivity, and the influence of hydrogen bonding will be presented.
This research was supported by the US NIH grants ES 04026, ES 04184, DK 51308 and NSF 9512622
Converted to HTML by F. Holger Försterling March 2001
