摘要
蛋白质翻译后修饰产生的3-氯酪氨酸(3-Cl-Tyr)与多种疾病相关,包括帕金森病、哮喘、动脉粥样硬化等.在动脉粥样硬化患者中发现AopA1 192位酪氨酸有高水平的氯化,显示此种修饰可能会促进病变.为了研究酪氨酸氯代对蛋白质功能的调控作用,我们发展了将3-Cl-Tyr定点特异插入到蛋白质中的方法.因为3-Cl-Tyr酚羟基上的质子比酪氨酸(Tyr)更容易解离,具有更低的pKa,在绿色荧光蛋白GFP及其突变体,以及光转化荧光蛋白mEOS2荧光活性中心中分别用3-Cl-Tyr取代Tyr,使得GFP发色基团的pKa降低到4.7,并且具有与EGFP相似的量子产率,使mEOS2发色基团的pKa降低到4.2.这样使得荧光蛋白的发色基团在酸性条件下仍然能以去质子化形式存在,在500 nm以上仍然具有较强吸收,避免了用400 nm左右激光激发及其对细胞及细胞器造成的光损伤.这种新型的荧光蛋白突变体将适用于溶酶体、吞噬酶体等酸性细胞器.
Posttranslational chlorination of tyrosine residues in proteins produce 3-chlorotyrosine（3-Cl-Tyr）,which is associated with several diseases,including Alzheimer＇s disease,asthma,atherosclerosis and acute myocardial infarction.High level of 3-chlorotyrosine has been found in ApoA1 protein in atherosclerosis patients,indicating that it may play important role in disease.Here we report a new method to facilitate the site-specific incorporation of 3-chlorotyrosine into proteins at specific sites.Such a new method may be very useful to probe the regulatory role of tyrosine chlorination in protein function.Compared to tyrosine（Tyr）,3-Cl-Tyr has lower pKa.We replaced the green fluorescent protein（GFP） and photoactivatable protein mEOS2 chromophore Tyr（Tyr66 in GFP） by 3-Cl-Tyr,lowering the chromophore pKa to 4.2 and 4.7,respectively.These mutant fluorescent proteins with lower pKa may be advantageous for labeling proteins in acidic organelles such as lysosome and phagosome.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2012年第4期378-387,共10页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(30870592
90913022)资助项目~~
