光谱及分子探针方法研究番茄Metacaspase蛋白与Ca^(2+)的相互作用  

Research on the Tomato Metacaspase Protein Interactions with Ca^(2+) by Spectroscopy and Molecular Probe

作　　者：温帅[1] 马营轩 吴坤胜[1] 崔金田 罗云波[1] 曲桂芹[1] 

机构地区：[1]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《光谱学与光谱分析》2015年第6期1643-1648,共6页Spectroscopy and Spectral Analysis

基　　金：国家(973计划)项目(2013CB127104);现代农业产业技术研究体系项目(ycytx-30-zy-05)资助

摘　　要：Metacaspases(MCPs)是发现于植物、真菌、原生动物体内的一种结构上类似多细胞动物Caspase的半胱氨酸蛋白酶家族,其大多数成员的激活依赖于钙离子,但钙离子如何影响MCPs的激活机制有待深入研究。借助圆二色谱技术、Terbium/Stains-all探针技术以及荧光光谱技术,以番茄Ⅱ型Metacaspase(LeMCA1)重要位点突变的三种体外原核表达重组蛋白,包括LeMCA1C139A(活性催化位点突变体)、LeMCA1K223G(自降解位点突变体)以及预测的Ca2+结合位点突变体LeMCA1D116A/D117A为研究材料,探究了MCPs与Ca2+相互作用机制。实验结果表明,Ca2+与LeMCA1之间既不存在强烈的结合作用,也不影响蛋白的二级结构,但Ca2+通过相互作用改变LeMCA1的三级结构来实现对LeMCA1的酶原激活过程。其中,LeMCA1蛋白的Asp-116,Asp-117氨基酸残基作为预测的与Ca2+作用的重要位点,其缺失将导致蛋白与Ca2+相互作用能力下降。利用圆二色谱、荧光光谱结合离子探针技术研究了典型茄科植物番茄中Ca2+与LeMCA1的相互作用特性,结合之前同源序列比对、位点突变结果确定了LeMCA1中的Asp-116,Asp-117氨基酸残基影响着Ca2+与蛋白质的相互作用。该结果对后续LeMCA1的生化特性及晶体结构的解析研究有着重要的参考价值。Metacaspases are cysteine-dependent proteases found in protozoa,fungi and plants and are distantly related to metazoan caspases.Most of MCPs activation are the calcium dependent,but the mechanisms are still unknown.Based on the techniques of CD spectroscopy,fluorescence spectroscopy,and Terbium Stains-all probe,we selected three purified recombinant proteins from key residues mutated in tomato metacaspase（LeMCA1）,including conserved catalytic site（C139A）mutant,N-sequenced cleaved site（K223G）mutant and the predicted Ca^2＋binding sites（D116A/D117A）mutant,to explore the interaction mechanism of LeMCA1 and Ca^2＋.CD spectroscopy and Stains-all probe results suggested that the intense binding does not exist between LeMCA1 and Ca^2＋as well as Ca^2＋has little effect on the secondary structure of LeMCA1.However,fluorescence spectroscopy and Tb3＋probe results showed that Ca^2＋-induced the changes occur in the tertiary structure of LeMCA1,which contributes to the activation of zymogen.In addition,predicted Ca^2＋binding residues,Asp-116 and Asp117,are the key sites responsible for the Ca^2＋interaction with LeMCA1,and the loss of these two residues resulted in decreased interaction.Our data firstly provided insight on the mechanism of the interaction between Ca^2＋and recombinant purified Solanaceae type Ⅱ metacaspase by spectroscopy and molecular probe techniques.Combined the results we got before from sequence-alignment and sitesmutation,the key residues Asp-116 and Asp117affect the Ca^2＋-induced the changes of LeMCA1 tertiary structure.Our data provided information for the further biochemical and crystal assays of LeMCA1.

关 键 词：番茄Metacaspase CA2+ 圆二色谱 荧光光谱 Ca2+探针 

分 类 号：O657.3[理学—分析化学]