摘要
目的用定点自旋标记-电子顺磁共振(SDSL-EPR)技术检测自噬相关蛋白LC3B功能结构域上特定氨基酸位点的运动特性。方法采用序列重叠延伸PCR(SOE-PCR)法对LC3B蛋白NHD结构域α1螺旋Q9位点、ULD结构域α3螺旋N59位点以及口袋结构内部β1折叠135位点引入半胱氨酸(cysteamine,Cys)突变,表达纯化获得LC3B突变体蛋白;甲烷硫代磺酸(2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate,MTSL)自旋标记后进行电子顺磁共振(EPR)检测,通过计算EPR波谱旋转相关时间τc,分析不同氨基酸位点的运动特性。结果构建了LC3B突变体基因的原核表达载体pET-22b-LC3B,实现了蛋白的可溶性高效表达;Q9C和N59C位点EPR波谱呈现双成分叠加谱,Q9C位点不同运动成分的τc值分别为1和3.8 ns,N59C位点为1和3.4 ns;I35C位点为三成分叠加谱,其τc值分别为0.7、2.9和8.1 ns。结论存在于特定氨基酸位点的运动性差别,预示着溶液状态下LC3B蛋白各功能结构域的同一氨基酸位点处可能存在不同的构象特征。
Objective To analyze the mobility of amino acid residue sites in the autophagy related protein-microtubuleassociated protein 1 light chain 3 B(LC3 B)by site directed spin labeling-electron paramagnetic resonance(SDSL-EPR)technology.Methods Three residue sites,Q9 site in theα1 helix of N-terminal helical domain(NHD)domain,N59 site in the a3 helix of C-terminal ubiquitin-like domain(ULD)domain and 135 site in the sheet of pocket structure of LC3 B were mutated to cysteamine(Cys)by using sequence overlapped extension PCR(SOE-PCR).The LC3 B mutants were then expressed and purified and spin labeled with 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate(MTSL)for EPR experiment.The rotational correlation timeτc of EPR spectra was calculated in order to study the characteristics of the mobility of selected amino acid sites.Results The prokaryotic expression vectors pET-22 b-LC3 B were successfully constructed,by which the LC3 B mutants were efficiently expressed in the form of soluble protein.The EPR spectra of Q9 C and N59 C sites showed a two-component superimposed spectrum.Theτc of Q9 C site was 1 and 3.8 ns,and theτc of N59 C site was 1 and 3.4 ns.The spectra of I35 C site showed a three-component superimposed spectrum,of which theτc values were 0.7,2.9 and 8.1 ns.Conclusion The difference of mobility in amino acid residues suggests that there can exist different conformational characteristics in selected sites of the functional domain in LC3 B in solution.
作者
赵曦
董国福
马蕾
郭俊旺
吴可
ZHAO Xi;DONG Guo fu;MA Lei;GUO Jun wang;WU Ke(Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处
《军事医学》
CAS
北大核心
2019年第9期664-668,共5页
Military Medical Sciences
基金
国家自然科学基金项目(31800053).
