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 logβ’_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 logβ’_{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 logβ’ 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

中文翻译：

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通过与 DNA 的相互作用增强 Cys2His2 型锌指蛋白的锌结合

摘要

锌指蛋白特异性识别 DNA 序列，因此在生物体中起着至关重要的作用。在这项研究中，Zn(II)- 和 1MEY# 的 DNA 结合，一种由三个指单元组成的人工锌指蛋白，通过多种方法进行了表征。应用荧光、圆二色性和等温量热滴定来确定锌指蛋白的准确稳定常数。假设所有三个锌指亚基的行为相同，获得的 Zn(II) 结合热力学数据为ΔH_结合位点 = − (23.5 − 28.0) kcal/mol（取决于半胱氨酸的应用质子化状态）和 log β '_{酸碱度 7.4} = 12.2 ± 0.1，与 CP1 共识锌指肽相似。蛋白质的特异性 DNA 结合可以通过 log β ' _{pH 7.4}  = 8.20 ± 0.08 来表征，这与天然锌指蛋白（Sp1、WT1、TFIIIA）对 DNA 的亲和力相当。该值比为半特异性或非特异性 DNA 结合确定的值高~ 1.9 log β ' 单位。竞争性圆二色性和电泳迁移率变动测量表明，在其目标 DNA 序列存在的情况下，1MEY# 蛋白的 Zn(II) 结合条件稳定性常数特征增加了 3.4 个数量级。

图形概要