通过分子内相互作用控制bHLH蛋白的DNA结合特异性。

Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions.

作者信息

Turner Elizebeth C, Cureton Charlotte H, Weston Chris J, Smart Oliver S, Allemann Rudolf K

机构信息

School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

出版信息

Chem Biol. 2004 Jan;11(1):69-77. doi: 10.1016/j.chembiol.2003.12.015.

DOI:10.1016/j.chembiol.2003.12.015

PMID:15112996

Abstract

Reversible control of the conformation of proteins was employed to probe the relationship between flexibility and specificity of the basic helix-loop-helix protein MyoD. A fusion protein (apaMyoD) was designed where the basic DNA binding helix of MyoD was stablized by an amino-terminal extension with a sequence derived from the bee venom peptide apamin. The disulfide-stabilized helix from apamin served as a nucleus for a helix that extended for a further ten residues, thereby holding apaMyoD's DNA recognition helix in a predominantly alpha-helical conformation. The thermal stability of the DNA complexes of apaMyoD was increased by 13 degrees C relative to MyoD-bHLH. Measurements of the fluorescence anisotropy change on DNA binding indicated that apaMyoD bound to E-box-containing DNA sequences with enhanced affinity relative to MyoD-bHLH. Consequently, the DNA binding specificity of apaMyoD was increased 10-fold.

摘要

利用蛋白质构象的可逆控制来探究碱性螺旋-环-螺旋蛋白MyoD的柔韧性与特异性之间的关系。设计了一种融合蛋白（apaMyoD），其中MyoD的碱性DNA结合螺旋通过氨基末端延伸得以稳定，该延伸序列源自蜂毒肽蜂毒明肽。来自蜂毒明肽的二硫键稳定螺旋作为进一步延伸十个残基的螺旋的核心，从而使apaMyoD的DNA识别螺旋保持在主要为α螺旋的构象中。相对于MyoD-bHLH，apaMyoD的DNA复合物的热稳定性提高了13摄氏度。对DNA结合时荧光各向异性变化的测量表明，相对于MyoD-bHLH，apaMyoD与含E-box的DNA序列结合时亲和力增强。因此，apaMyoD的DNA结合特异性提高了10倍。