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.
Laity
John H
JH
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO, USA.
Feng
Linda S
LS
D000818:Animals; D002151:Calorimetry; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics*; D000097809:Transcription Factor MTF-1
D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1; C103264:ZAP1 protein, S cerevisiae