Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA

IF 2.7 3区 化学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Bálint Hajdu, Éva Hunyadi-Gulyás, Kohsuke Kato, Atsushi Kawaguchi, Kyosuke Nagata, Béla Gyurcsik
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引用次数: 1

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 ΔHbinding site =  − (23.5 − 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logβpH 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 logβpH 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.

Abstract Image

cys2his2型锌指蛋白的锌结合通过与DNA的相互作用而增强
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。本研究通过多种方法表征了由三个指单元组成的人造锌指蛋白1mey#的Zn(II)-与dna的结合。采用荧光法、圆二色法和等温量热滴定法测定锌指蛋白的准确稳定性常数。假设所有三个锌指亚基的行为相同,得到的Zn(II)结合的热力学数据为ΔHbinding位点=−(23.5−28.0)kcal/mol(取决于半胱氨酸的质子化状态)和logβ ' pH 7.4 = 12.2±0.1,与CP1共识锌指肽相似。该蛋白与DNA的特异性结合可表征为logβ′pH 7.4 = 8.20±0.08,与天然锌指蛋白(Sp1, WT1, TFIIIA)对DNA的亲和力相当。该值比半特异性或非特异性DNA结合的值高~ 1.9 logβ’单位。竞争性圆二色性和电泳迁移率位移测量表明,在其靶DNA序列存在的情况下,Zn(II)结合1mey#蛋白的条件稳定常数特性增加了3.4个数量级。
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来源期刊
JBIC Journal of Biological Inorganic Chemistry
JBIC Journal of Biological Inorganic Chemistry 化学-生化与分子生物学
CiteScore
5.90
自引率
3.30%
发文量
49
审稿时长
3 months
期刊介绍: Biological inorganic chemistry is a growing field of science that embraces the principles of biology and inorganic chemistry and impacts other fields ranging from medicine to the environment. JBIC (Journal of Biological Inorganic Chemistry) seeks to promote this field internationally. The Journal is primarily concerned with advances in understanding the role of metal ions within a biological matrix—be it a protein, DNA/RNA, or a cell, as well as appropriate model studies. Manuscripts describing high-quality original research on the above topics in English are invited for submission to this Journal. The Journal publishes original articles, minireviews, and commentaries on debated issues.
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