Preparation and zinc-binding properties of multi-fingered zinc-sensing domains

Cys(2)His(2) zinc finger proteins (ZFPs) adopt a highly conserved betabetaalpha-fold, which is stabilized by tetrahedral coordination of a central zinc ion by two cysteine and two histidine residues. Although the function of most zinc fingers is sequence-specific DNA binding, other roles such as zinc sensing have been identified. Here, methods are described to produce micromole quantities of zinc-sensing zinc finger domains from the Zap1 (two zinc fingers) and MTF-1 (six zinc fingers) transcription factors. Procedures are outlined to isolate recombinant zinc finger proteins from a bacterial expression system that generates both soluble intracellular (Zap1) and insoluble inclusion body (MTF-1) forms. Isolated proteins are reduced and subsequently HPLC purified at low pH and lyophilized, which generates proteins that are ideal for zinc-binding studies or metal substitution studies and stable during long-term storage. NMR and calorimetric methods are described to measure relative and individual zinc ion affinities in multi-fingered proteins, which is an essential step in unraveling the zinc-sensing mechanism of MTF-1 and Zap1.