Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA
Bálint Hajdu, Éva Hunyadi‐Gulyás, Kohsuke Kato, Atsushi Kawaguchi, Kyosuke Nagata, Béla Gyurcsik
Abstract
Abstract Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔH binding site = − (23.5 − 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and log β ’ pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by log β ’ pH 7.4 = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 log β ’ unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence. Graphical abstract