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重组别藻蓝蛋白的超快能量传递过程

甄张赫 朱锐丹 秦松 陈海龙 蒲洋 翁羽翔 李文军

甄张赫,朱锐丹,秦松,等. 重组别藻蓝蛋白的超快能量传递过程[J]. 海洋学报,2021,43(9):115–125 doi: 10.12284/hyxb2021120
引用本文: 甄张赫,朱锐丹,秦松,等. 重组别藻蓝蛋白的超快能量传递过程[J]. 海洋学报,2021,43(9):115–125 doi: 10.12284/hyxb2021120
Zhen Zhanghe,Zhu Ruidan,Qin Song, et al. Ultra-fast energy transfer process of recombinant allophycocyanin[J]. Haiyang Xuebao,2021, 43(9):115–125 doi: 10.12284/hyxb2021120
Citation: Zhen Zhanghe,Zhu Ruidan,Qin Song, et al. Ultra-fast energy transfer process of recombinant allophycocyanin[J]. Haiyang Xuebao,2021, 43(9):115–125 doi: 10.12284/hyxb2021120

重组别藻蓝蛋白的超快能量传递过程

doi: 10.12284/hyxb2021120
基金项目: 国家自然科学基金(41906109);中国科学院前沿科学重点研究项目(QYZDJ-SSW-SYS017)
详细信息
    作者简介:

    甄张赫(1992—),女,辽宁省盖平县人,博士研究生,研究藻类捕光蛋白结构及其功能机制。E-mail:zhzhen@yic.ac.cn

    朱锐丹(1992—),男,广东省揭阳市人,博士研究生,研究光合捕光蛋白的超快传能机制。E-mail:zhuruidan@iphy.ac.cn

    通讯作者:

    翁羽翔,研究员,主要从事光合捕光天线蛋白的超快传能和动态调控机制的研究。E-mail:yxweng@aphy.iphy.ac.cn

    李文军,副研究员,主要从事藻类功能蛋白的结构功能和应用研究相关工作。E-mail:wjli@yic.ac.cn

  • 中图分类号: Q949.2; Q946.1

Ultra-fast energy transfer process of recombinant allophycocyanin

  • 摘要: 藻胆体是红、蓝藻特有的光合作用捕光天线复合物。别藻蓝蛋白(APC)是组成藻胆体高效能量传递的核心结构的主要成分。本文以基因重组的别藻蓝蛋白(rAPC)的单体和三聚体为材料,通过稳态光谱、圆二色光谱以及超快时间分辨光谱研究了rAPC的结构构象和能量传递过程。结果表明,rAPC在测试条件下能保持和天然APC一致的光谱特性和活性构象;rAPC单体组装成三聚体后,其α84PCB和β84PCB可以组成激子色素对,通过激子分裂提高三聚体的能量传递效率;超快时间分辨光谱结果显示,在rAPC三聚体中,能量从620 nm传至650 nm的时间为300~600 fs,同时存在着19 fs的激子态的电子退相干过程。这些结果为揭示藻胆体的高效能量传递机制提供了数据基础。
  • 图  1  别藻蓝蛋白三聚体晶体结构图

    结构信息下载于PDB数据库(http://www1.rcsb.org/),PDB码为4F0U;灰色螺旋为别藻蓝蛋白的螺旋结构,灰色杆代表色素分子;粉色螺旋和黄色螺旋组成了别藻蓝蛋白的单体,其中黄色螺旋为别藻蓝蛋白α亚基,粉色螺旋为别藻蓝蛋白β亚基;绿色杆代表α84PCB;粉色杆代表β84PCB

    Fig.  1  Allophycocyanin trimer crystal structure

    The structure data is downloaded from the PDB database (http://www1.rcsb.org/), PDB code is 4F0U; the gray spiral represents spiral structure of allophycocyani; the gray sticks represent pigment molecules; the pink spiral (β subunit) and the yellow spiral (α subunit) constitute the allophycocyanin monomer; green sticks represent α84PCB; pink sticks represent β84PCB

    图  2  诱导后菌体的稳态光谱及荧光性质变化

    a. 诱导前和诱导后的菌体在蓝光和绿光激发下的荧光发射图片;b. 诱导后绿色菌体的稳态吸收光谱和荧光发射光谱(Ex615 nm

    Fig.  2  Steady-state spectra and fluorescence properties of induced bacteria

    a. Fluorescence emission pictures of the bacteria before and after induction (green light excitation); b. steady-state absorption and fluorescence emission spectra (Ex615 nm) of the blue-green bacteria after induction

    图  3  rAPC三聚体纯化及亚基鉴定

    a. rAPC蔗糖密度分离结果,上层rAPC单体,下层rAPC三聚体;b. rAPC三聚体;c. rAPC三聚体SDS-PAGE蛋白电泳图

    Fig.  3  Purification and composition identification of rAPC trimer

    a. rAPC sucrose density separation result, upper rAPC monomer, lower rAPC trimer; b. rAPC trimer; c. SDS-PAGE protein electrophoresis of rAPC trimer

    图  4  rAPC单体和三聚体的稳态吸收光谱及荧光发射光谱

    a. rAPC单体和三聚体的稳态吸收光谱,为了方便对比,进行了0~1区间的归一化处理,粉色虚线为单体吸收光谱的二阶导数图,灰色虚线为三聚体吸收光谱的二阶导数图;b. rAPC单体和三聚体的稳态荧光发射光谱,为了方便对比进行了0~100区间的归一化处理

    Fig.  4  Steady-state absorption spectra and fluorescence spectra of rAPC trimer

    a. The steady-state absorption spectra of rAPC monomer and trimer are normalized in the range of 0‒1 for the convenience of comparison, the pink dotted line is the second derivative diagram of the monomer absorption spectrum, and the gray dotted line is the second derivative diagram of the trimer absorption spectrum; b. the steady-state fluorescence emission spectra of rAPC monomer and trimer are normalized in the interval of 0‒100 for the convenience of comparison

    图  5  rAPC单体和三聚体的CD光谱

    a. rAPC单体和三聚体CD的紫外区光谱;b. rAPC单体和三聚体CD的可见光区光谱

    Fig.  5  CD spectra of rAPC monomer and trimer

    a. Circular dichroism spectra of rAPC monomer and trimer in the ultraviolet region; b. circular dichroism spectra of rAPC monomer and trimer in the visible region

    图  6  NOPA脉冲覆盖范围及rAPC单体和三聚体稳态吸收光谱

    NOPA-1. rAPC三聚体探测脉冲覆盖范围;NOPA-2. rAPC单体探测脉冲覆盖范围

    Fig.  6  Coverage of NOPA pulse and steady-state absorption spectra of rAPC trimer

    NOPA-1. rAPC trimer detection pulse coverage; NOPA-2. rAPC monomer detection pulse coverage

    图  7  rAPC单体及三聚体的激发能量依赖

    a. rAPC单体激发能量依赖线性拟合;b. rAPC三聚体激发能量依赖线性拟合

    Fig.  7  Excitation energy dependence of rAPC monomer and trimer

    a. The linear fitting of excitation energy in rAPC monomer; b. the linear fitting of excitation energy in rAPC trimer

    图  8  rAPC三聚体及单体的瞬态探测

    a. rAPC单体和三聚体在620 nm处的能量传递动力学拟合曲线;b. rAPC三聚体在620 nm及656 nm处的能量传递动力学拟合曲线

    Fig.  8  Transients detected of rAPC trimer and monomer

    a. Fitting curve of energy transfer kinetics of rAPC monomer and trimer at 620 nm; b. fitting curve of energy transfer kinetics of rAPC trimer at 620 nm and 656 nm

    图  9  rAPC单体及三聚体不同时间下的瞬态光谱变化

    a. rAPC单体在不同时间分辨率下的瞬态光谱变化;b. rAPC三聚体在不同时间分辨率下的瞬态光谱变化

    Fig.  9  Energy transfer dynamics of rAPC monomer and trimer at several delay times

    a. Transient spectral change of rAPC monomers at different time resolutions; b. transient spectral change of rAPC trimers at different time resolutions

    表  1  rAPC单体和三聚体瞬态吸收动力学拟合常数

    Tab.  1  The fitting constants of rAPC trimer transient absorption dynamics

    样品波长时间寿命常数1时间寿命常数2时间寿命常数3
    rAPC单体620 nm(57±10)fs(7.4±4.8)ps400 ps
    rAPC三聚体620 nm(19±7)fs(630 ±100)fs400 ps
    rAPC三聚体656 nm(470 ±100)fs400 ps
      注:−代表未拟合到时间寿命常数。
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  • 收稿日期:  2021-02-19
  • 修回日期:  2021-04-13
  • 网络出版日期:  2021-05-28
  • 刊出日期:  2021-09-06

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