Department of Biomedical Science, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK ; IBLS, University of Glasgow, Glasgow G12 8QQ, UK.
FEBS Open Bio. 2012 Jan 21;2:6-11. doi: 10.1016/j.fob.2012.01.001. Print 2012.
Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F-actin have revealed a number of different conformations with calponin homology domains in 'open' and 'closed' positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F-actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F-actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino-terminal utrophin actin binding domain is in an open conformation in solution and when bound to F-actin.
结构分析表明，由串联钙调蛋白同源结构域组成的肌动蛋白结合区，无论是单独存在还是与 F-肌动蛋白结合，都呈现出多种不同的构象，钙调蛋白同源结构域呈现“开放”和“关闭”两种状态。为了解决这些问题，我们研究了处于开放和关闭构象的 utrophin 肌动蛋白结合域的特性，以验证其与 F-肌动蛋白结合时的构象。通过在每个钙调蛋白同源结构域中使用工程化半胱氨酸残基将肌动蛋白结合域锁定在关闭构象中，降低了与 F-肌动蛋白的亲和力，而不影响计量关系。此外，差示扫描量热法实验表明，与肌动蛋白结合时的熔点降低。这些数据表明，氨基末端 utrophin 肌动蛋白结合域在溶液中呈开放构象，与 F-肌动蛋白结合时也是如此。
