NMR spectroscopic studies on metallothioneins

The proposed project aims at investigating a class of metal-binding proteins called metallothioneins (MTs) and their interactions with other compounds by nuclear magnetic resonance spectroscopy. Metallothioneins have been known for over 40 years, but no clear picture about their biological function(s) evolved, although they are believed to play a key role in the homeostasis of zinc and copper and in the detoxification of heavy metals like cadmium and mercury. With the studies proposed in this application we expect to gain more insight into two recent findings which have attracted much attention to MTs. Both deal with the metal release from the N-terminal domain by either an oxido-reductive mechanisms mediated through the oxidized/reduced glutathione redox couple or nitric oxide. The binding of glutathione to the N-terminal domain will be studied by paramagnetic surface probes and the interplay between GSH/GGSG---NO---MT monitored structurally by NMR spectroscopy and through the release of zinc and/or cadmium. Thereby, special emphasis is placed on the suggested physiological role of MTs at inflammatory sites. For a better understanding of these processes a better defined structure of the N-terminal domain as well as information about the interdomain orientation of MTs in solution is highly desirable. Towards this goal, paramagnetic surface probes will be used to obtain additional NMR constraints for the solution structure which usually suffers from the limited number of NOEs due to almost missing regular secondary structural elements. The interdomain orientation of metallothionein in solution will be probed via the orientation dependence of N-15 NMR relaxation times.