天然同位素丰度野生型酵母细胞色素c构象变化的核磁共振检测

方仲佩; 孙鹏; 王倩文; 张亮; 刘买利; 张许

doi:10.11938/cjmr20192740

波谱学杂志 >

2019 , Vol. 36 >Issue 4: 481 - 489

DOI: https://doi.org/10.11938/cjmr20192740

研究论文

天然同位素丰度野生型酵母细胞色素c构象变化的核磁共振检测

方仲佩 ,
孙鹏 ,
王倩文 ,
张亮 ,
刘买利 ,
张许

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1. 波谱与原子分子物理国家重点实验室, 武汉磁共振中心(中国科学院武汉物理与数学研究所), 湖北武汉 430071;
2. 中国科学院大学, 北京 100049

收稿日期: 2019-04-18

网络出版日期: 2019-05-30

基金资助

国家重点研发计划（2017YFA0505400）.

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Conformational Change of Wild Type Cytochrome c Characterized by NMR Spectroscopy at Natural Isotropic Abundance

FANG Zhong-pei ,
SUN Peng ,
WANG Qian-wen ,
ZHANG Liang ,
LIU Mai-li ,
ZHANG Xu

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1. State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan(Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences), Wuhan 430071, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China

Received date: 2019-04-18

Online published: 2019-05-30

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摘要

细胞色素c是一个重要的多功能蛋白，它在呼吸链及细胞凋亡中均发挥着重要作用.细胞色素c的构象变化与其功能密切相关，表征细胞色素c的构象变化对于明确其相关功能的分子机制至关重要.核磁共振（NMR）是近原位环境下表征蛋白构象的重要工具，但通常需要使用¹³C、¹⁵N等同位素.由于野生型细胞色素c存在翻译后修饰，故其同位素标记十分困难.本文尝试使用¹H-¹³C HSQC技术来表征提纯后的天然同位素丰度的野生型细胞色素c的构象变化.结果显示该方法能在2 h内检测到大多数甲基的信号，且化学位移变化明显的甲基与其构象变化一致.这表明该方法有助于研究天然丰度或翻译后修饰蛋白的构象变化.

关键词： 核磁共振(NMR); 构象变化; ¹H-¹³C HSQC; 细胞色素c

本文引用格式

方仲佩 , 孙鹏 , 王倩文 , 张亮 , 刘买利 , 张许 . 天然同位素丰度野生型酵母细胞色素c构象变化的核磁共振检测[J]. 波谱学杂志, 2019 , 36(4) : 481 -489 . DOI: 10.11938/cjmr20192740

Abstract

Cytochrome c is an important multifunctional protein, which plays important roles in the respiratory chain and cell apoptosis. Characterization of conformational changes of cytochrome c is essential to elucidate the molecular mechanism underlying its functions. Isotope-labeled proteins are usually needed for nuclear magnetic resonance (NMR)-based protein structure elucidation. However, post-translational modification of cytochrome c makes it difficult to be labelled by isotopes. In this work, ¹H-¹³C HSQC NMR experiment was used to characterize the conformational changes of naturally purified wild-type cytochrome c in the redox process, focusing on the side chain methyl groups. It was observed that this method could detect the signals of most methyl groups within 2 h, and the methyl groups detected with apparent chemical shift change were coincide with its conformation change. The results indicated that it is possible to use the method proposed to study the conformational changes of proteins at natural abundance or post-translational modified proteins.

Key words： nuclear magnetic resonance (NMR); conformational change; ¹H-¹³C HSQC; cytochrome c

参考文献

[1] BANCI L, BERTINI I, GRAY H B, et al. Solution structure of oxidized horse heart cytochrome c[J]. Biochem, 1997, 36(32):9867-9877.
[2] FETROW J S, BAXTER S M. Assignment of N-15 chemical shifts and N-15 relaxation measurements for oxidized and reduced iso-1-cytochrome c[J]. Biochem, 1999, 38(14):4480-4492.
[3] KALPAGE H A, BAZYLIANSKA V, RECANATI M A, et al. Tissue-specific regulation of cytochrome c by post-translational modifications:respiration, the mitochondrial membrane potential, ROS, and apoptosis[J]. Faseb J, 2019, 33(2):1540-1553.
[4] LIU X S, KIM C N, YANG J, et al. Induction of apoptotic program in cell-free extracts:Requirement for dATP and cytochrome c[J]. Cell, 1996, 86(1):147-157.
[5] ZAIDI S, HASSAN M I, ISLAM A, et al. The role of key residues in structure, function, and stability of cytochrome-c[J]. Cell Mol Life Sci, 2014, 71(2):229-255.
[6] ALVAREZ-PAGGI D, HANNIBAL L, CASTRO M A, et al. Multifunctional cytochrome c:Learning new tricks from an old dog[J]. Chem Rev, 2017, 117(21):13382-13460.
[7] OVIEDO-ROUCO S, CASTRO M A, ALVAREZ-PAGGI D, et al. The alkaline transition of cytochrome c revisited:Effects of electrostatic interactions and tyrosine nitration on the reaction dynamics[J]. Arch Biochem Biophys, 2019, 665:96-106.
[8] MUNEESWARAN G, KARTHEESWARAN S, MUTHUKUMAR K, et al. Temperature-dependent conformational dynamics of cytochrome c:Implications in apoptosis[J]. J Mol Graph Model, 2018, 79140-148.
[9] DONG A, BROWN C, BAI S, et al. Acid-enhanced conformation changes of yeast cytochrome c coated onto gold nanoparticles, a FT-IR spectroscopic analysis[J]. Int J Biol Macromol, 2018, 112591-597.
[10] DANIELSON T A, STINE J M, DAR T A, et al. Effect of an imposed contact on secondary structure in the denatured state of yeast iso-1-cytochrome c[J]. Biochem, 2017, 56(51):6662-6676.
[11] BAISTROCCHI P, BANCI L, BERTINI I, et al. Three-dimensional solution structure of Saccharomyces cerevisiae reduced iso-1-cytochrome c[J]. Biochem, 1996, 35(43):13788-13796.
[12] IMAI M, SAIO T, KUMETA H, et al. Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c[J]. Biochem Biophys Res Commun, 2016, 469(4):978-984
[13] BARAYEU U, LANGE M, MENDEZ L, et al. Cytochrome c autocatalyzed carbonylation in the presence of hydrogen peroxide and cardiolipins[J]. J Biol Chem, 2019, 294(6):1816-1830.
[14] MOHAMMADYANI D, YANAMALA N, SAMHAN-ARIAS A K, et al. Structural characterization of cardiolipin-driven activation of cytochrome c into a peroxidase and membrane perturbation[J]. BBA-Biomembranes, 2018, 1860(5):1057-1068.
[15] AMACHER J F, ZHONG F, LISI G P, et al. A compact structure of cytochrome c trapped in a lysine-ligated state:loop refolding and functional implications of a conformational switch[J]. J Am Chem Soc, 2015, 137(26):8435-8449.
[16] HANSKE J, TOFFEY J R, MORENZ A M, et al. Conformational properties of cardiolipin-bound cytochrome c[J]. Proc Natl Acad Sci USA, 2012, 109(1):125-130.
[17] KOBAYASHI H, NAGAO S, HIROTA S. Characterization of the cytochrome c membrane-binding site using cardiolipin-containing bicelles with NMR[J]. Angew Chem Int Ed Engl, 2016, 55(45):14019-14022.
[18] QI H, JIANG Y, YIN Z Y, et al. Optimal pathways for the assembly of the Apaf-1.cytochrome c complex into apoptosome[J]. Phys Chem Chem Phys, 2018, 20(3):1964-1973.
[19] JIN J P. Protein structure, function, and regulation in biological movement[J]. Arch Biochem Biophys, 2006, 456(2):99-101.
[20] KAY L E. NMR studies of protein structure and dynamics-a look backwards and forwards[J]. J Magn Reson, 2011, 213(2):492-494.
[21] LI H, KAMATARI Y O, KITAHARA R, et al. High-pressure nmr for studying protein structure and dynamics[J]. Chinese J Magn Reson, 2016, 33(1):1-26. 李华, KAMATARI Y O, KITAHARA R, 等. 高压NMR在蛋白质结构和动力学研究中的应用[J]. 波谱学杂志, 2016, 33(1):1-26.
[22] RAN M L, QIN L Y, TANG C, et al. Regulation of inter-protein interactions between ubiquitin and ubiquitin-associated domains in Rad23A/ubiquilin-1 by phosphorylation[J]. Chinese J Magn Reson, 2019, 36(1):15-22. 冉梦琳, 覃凌云, 唐淳, 等. 磷酸化调控泛素单体与Rad23A/Ubiquilin-1中泛素结合域互作的检测[J]. 波谱学杂志, 2019, 36(1):15-22.
[23] MORAR A S, KAKOURAS D, YOUNG G B, et al. Expression of N-15-labeled eukaryotic cytochrome c in Escherichia coli[J]. J Biol Inorg Chem, 1999, 4(2):220-222.
[24] ARBOGAST L W, DELAGLIO F, TOLMAN J R, et al. Selective suppression of excipient signals in 2D H-1-C-13 methyl spectra of biopharmaceutical products[J]. J Biomol NMR, 2018, 72(3/4):149-161.
[25] LAN W X, WANG Z H, YANG Z Z, et al. Conformational toggling of yeast iso-1-cytochrome c in the oxidized and reduced states[J]. Plos One, 2011, 6(11).
[26] SZABO C M, SANDERS L K, LE H C, et al. Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and H-1,C-13 and N-15 chemical shift assignments by multidimensional NMR[J]. Febs Letters, 2000, 482(1/2):25-30.
[27] POLLOCK W B R, ROSELL F I, TWITCHETT M B, et al. Bacterial expression of a mitochondrial cytochrome c. Trimethylation of Lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition[J]. Biochem, 1998, 37(17):6124-6131.
[28] DELAGLIO F, GRZESIEK S, VUISTER G W, et al. NMR pipe-A multidimensional spectral processing system based on unix pipes[J]. J Biomol NMR, 1995, 6(3):277-293.
[29] LETT C M, ROSU-MYLES M D, FREY H E, et al. Rational design of a more stable yeast iso-1-cytochrome c[J]. BBA-Protein Struct M, 1999, 1432(1):40-48.
[30] SUN P, WANG Q W, YUAN B, et al. Monitoring alkaline transitions of yeast iso-1 cytochrome c at natural isotopic abundance using trimethyllysine as a native NMR probe[J]. Chem Comm, 2018, 54(89):12630-12633.

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