title: Spectroscopic determination of the thermodynamics of cobalt and zinc binding to GATA proteins.
abstract: Vertebrate GATA proteins regulate processes that are vital to development, and each possesses two tandem GATA finger domains: an N-terminal GATA finger and a C-terminal GATA finger. These GATA fingers require Zn(2+) to fold, to bind DNA recognition elements, and to regulate transcription. While the GATA-1 C-terminal finger is necessary and sufficient to bind to single GATA DNA sites, the N-terminal finger interacts with DNA such that the double finger unit (DF domain) has a binding and transactivation profile that is tuned by the DNA-binding site. Co(2+) was used as a spectroscopic probe in a series of competition titrations to determine the affinity of Co(2+) and Zn(2+) for the C-terminal finger from chicken GATA-1 and the double finger from human GATA-1 (referred to in this report as CF and DF). For CF, these experiments yielded K(b)(Co) = 1.0 (+/-1.3) x 10(7) M(-1) and K(b)(Zn) = 2.0 (+/-1.3) x 10(10) M(-1). For DF, these experiments yielded equilibrium constants for the process of two M(2+) binding to form M(2+)(2)-DF of beta(2)(Co) = 2.5 (+/-1.6) x 10(14) M(-2) and beta(2)(Zn) = 6.3 (+/-2.5) x 10(20) M(-2). The ZnS(4) coordination environment of Zn(2+)-bound CF was confirmed with X-ray absorption spectroscopy. A detailed analysis of these data suggests that the N-terminal and C-terminal fingers of DF act as independent and identical Zn(2+)-binding sites and each finger binds Zn(2+) with an affinity equivalent to that of CF.
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, USA.
authors.forename: Amy B
authors.initials: AB
authors.lastname: Ghering
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Shokes
authors.forename: Robert A
authors.initials: RA
authors.lastname: Scott
authors.forename: James G
authors.initials: JG
authors.lastname: Omichinski
authors.forename: Hilary A
authors.initials: HA
authors.lastname: Godwin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry / Q000379:methods*; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic; D015032:Zinc / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D007477:Ions; D008670:Metals; D014157:Transcription Factors; D003035:Cobalt; D004247:DNA; D015032:Zinc