The field of designing artificial metalloproteins has yet to effectively tackle the incorporation of multimetal clusters, which is a key component of natural metalloproteins, such as metallothioneins (MTs) and calmodulin. MT is a physiological, essential, cysteine-rich metalloprotein that binds to a variety of metals but is only known to form metal-thiolate clusters with Cd 2+ , Zn 2+ , and Cu + . Bismuth is a xenobiotic metal and a component of metallodrugs used to treat gastric ulcers and cancer, as well as an emerging metal used in industrial practices. Electrospray ionization mass spectrometry, UV−visible spectroscopy, and extended X-ray absorption fine structure spectroscopy were used to probe the Bi 3+ binding site structures in apo-MT3 (brain-located MT) at pH 7.4 and 2 and provide the complete set of binding affinities. We discovered the highly cooperative formation of a novel Bi 3+ species, Bi 2 MT3, under physiological conditions, where each Bi 3+ ion is coordinated by three cysteinyl thiolates, with one of the thiolates bridging between the two Bi 3+ ions. This cluster structure was associated with a strong visible region absorption band, which was disrupted by the addition of Zn 2+ and reversibly disrupted by acidification and increased temperature. This is the first reported presence of bridging cysteines for a xenobiotic metal in MT3 and the Bi 2 MT structure is the first Bi cluster found in a metalloprotein.

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Results from ESI-MS and stopped flow kinetics show that apo-MT protects from toxic metalation of apo-CA with Cd2+ due to the protein–protein interactions in solution.

Carbonic anhydrases (CAs) and metallothioneins (MTs) are both families of zinc metalloproteins central to life, however, they coordinate and interact with their Zn2+ ion cofactors in completely different ways. CAs and MTs are highly sensitive to the cellular environment and play key roles in maintaining cellular homeostasis. In addition, CAs and MTs have multiple isoforms with differentiated regulation. This review discusses current literature regarding these two families of metalloproteins in carcinogenesis, with a dialogue on the association of these two ubiquitous proteins in vitro in the context of metalation. Metalation of CA by Zn-MT and Cd-MT is described. Evidence for protein–protein interactions is introduced from changes in metalation profiles of MT from electrospray ionization mass spectrometry and the metalation rate from stopped-flow kinetics. The implications on cellular control of pH and metal donation is also discussed in the context of diseased states.

On behalf of the Clinical Investigator Trainee Association of Canada (CITAC) Board of Directors, I would like to extend an enthusiastic welcome to our new MD+ trainee members! I hope you soaked up all that summer had to offer and are in good back-to-school spirits. A new academic year is upon us, and opportunities abound for the Canadian physician scientist trainee community.