4 Summary and Conclusions
4.6 Future Directions
The novel ESI MS studies described in this thesis have opened many avenues for further study of MT and its roles in the homeostasis of essential metals, detoxification of toxic metals, and in protection against oxidative stress. Future studies should attempt to provide concrete evidence for the potential structures detailed in Figure 4.1. Crystal structures of Zn5-MT, Zn6-MT, and Zn7-MT would greatly impact the understanding of
the structure, and therefore, function of metallothioneins in the homeostasis of zinc. In addition, because terminal metal-thiolate bonds are thought to be shorter than bridging bonds, a difference in M-S bond length may be observed in XAFS studies of the terminally coordinated sites in Zn5-MT compared with the clustered binding sites in Zn7-
MT. With the understanding that obtaining a crystal structure is no small task, there may be other ways to investigate the stable Zn5-MT and its potential role in buffering zinc
concentrations within a cell. For example, the available cysteines can be counted using a covalent cysteine modifier, parabenzoquinone, which has been used previously to investigate the structure of apo-MT and the accessibility of partially metallated MT (130,
218). Modification of a cysteine with benzoquinone results in a mass shift of 108.09 Da from the mass of the protein and is thus easily identified in a deconvoluted mass spectrum. If the Zn2+ in Zn5-MT are indeed coordinated only by terminal cysteines, there
should be no sulfurs available to bind the benzoquinone. In fact, the use of benzoquinone as a modifier of available cysteines may aid in the elucidation of the various intermediate structures in the metallation pathway of MT.
Further investigation into the significance of the Zn5-rhMT intermediate species, through
interactions of Zn7-rhMT with Zn2+-dependent apo-enzymes, is also required. If
formation of a Zn5-species permits donation to apo-enzymes with binding affinities on
the order of 1011 M-1, Zn7-MT should readily donate two Zn2+. In addition, a sample of
100% Zn5-MT should readily accept two Zn2+ from Zn2+ transport proteins with lower
KFs than the sixth and seventh binding sites in βα-rhMT (on the order of 1010 M-1).
Now that the metallation mechanism for Zn2+ has been well characterized, with non- cooperative binding and a stable Zn5-rhMT intermediate, similar experiments are
required to decipher the binding mechanism of the other essential metal often bound by MT in vivo, Cu+. Copper coordination is different from that of Zn2+ (digonal or trigonal vs. tetrahedral) and therefore, the metallation pathway is not expected to be identical. It would be of interest to investigate the existence of an intermediate Cu-MT species that coordinates the Cu+ using only terminal cysteines. Investigation of the Cu+ metallation of
MT would be slightly more difficult than that of Zn2+ because not only will the twenty cysteinyl sulfurs oxidize, but so will the Cu+ if exposed to oxygen.
The two-domain structure of many metallothioneins is thought to allow the protein to function simultaneously in both Cu+ and Zn2+ homeostasis. Future metallation studies in which metallothionein is exposed to increasing amounts of both Cu+ and Zn2+ are needed to determine if the metallation pathway for metallothionein is different in the presence of both metals. In particular, a competition experiment in which the domain peptides compete for Cu+ and Zn2+ may give some indication of any domain preference for these metals. However, such an experiment may not be easily monitored by ESI MS because the masses of copper and zinc are quite close (63.55 and 65.38 g/mol, respectively).
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