基于19F化学标记磷酸化泛素的温度传感器开发

doi:10.11938/cjmr20202867

波谱学杂志 ›› 2021, Vol. 38 ›› Issue (2): 173-181.doi: 10.11938/cjmr20202867

基于¹⁹F化学标记磷酸化泛素的温度传感器开发

张志武^1,²,杨菊^1,²,聂泽锋^1,²,叶尚祥^1,^3,*(),董旭^1,*(),唐淳^1,^3,⁴

1. 中国科学院生物磁共振分析重点实验室, 波谱与原子分子物理国家重点实验室(中国科学院精密测量科学与技术创新研究院), 湖北武汉 430071
2. 中国科学院大学, 北京 100049
3. 华中科技大学武汉光电国家研究中心, 湖北武汉 430074
4. 北京分子科学国家研究中心, 北京大学化学与分子工程学院, 北京大学, 北京 100871

收稿日期:2020-10-16 出版日期:2021-06-05 发布日期:2020-11-10
通讯作者: 叶尚祥,董旭 E-mail:yeshangxiang@wipm.ac.cn;dongxu@wipm.ac.cn
基金资助:
国家重点研发计划(2018YFA0507700)

Development of a Temperature Senor Based on ¹⁹F-labeled Phosphorylated Ubiquitin

Zhi-wu ZHANG^1,²,Ju YANG^1,²,Ze-feng NIE^1,²,Shang-xiang YE^1,^3,*(),Xu DONG^1,*(),Chun TANG^1,^3,⁴

1. CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
3. Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China
4. Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

Received:2020-10-16 Online:2021-06-05 Published:2020-11-10
Contact: Shang-xiang YE,Xu DONG E-mail:yeshangxiang@wipm.ac.cn;dongxu@wipm.ac.cn

摘要/Abstract

摘要：

泛素是一种真核细胞信号分子，主要参与蛋白质降解和DNA修复等生命活动.泛素Ser65位被磷酸化之后，在溶液中呈现两个稳定的溶液构象，这两种构象的比例能够被pH调控，本研究利用NMR进一步发现它还受到温度影响.基于该发现，对磷酸化泛素进行了¹⁹F化学标记，利用¹⁹F NMR方法表征了不同温度下磷酸化泛素两种构象的比例，发现两者比例变化与温度之间的关系可以通过线性方程来描述，利用该方程可以通过构象比例计算样品内部温度，因此可以作为一种基于NMR检测的温度传感器.本文所开发的基于¹⁹F化学标记磷酸化泛素的温度传感器不仅能够作为体外样品温度检测的有力工具，还有望用于检测细胞内部的温度.从而有助于揭示生物学特性和功能.

关键词: 泛素, 磷酸化, 核磁共振, 温度传感器

Abstract:

Ubiquitin (Ub) is a signaling protein in eukaryotic cells, and it functions in the processes of protein degradation and DNA repairing. The published data indicated that the phosphorylation of Ub on Ser65 leads to two stable solution conformations, which can be regulated by pH. In the present project, it was found that the conformational ratio could also be modulated by temperature. Based on this feature, pUb was chemically labeled with a ¹⁹F probe for determination of conformational ratio under various temperatures using ¹⁹F NMR. We showed that the conformation ratio of pUb was linearly proportional to temperature gradient. Consequently, the temperature of a biological sample can be calculated with a given conformational ratio of pUb, and ¹⁹F-labeled pUb can function as a thermosensor. This thermosensor could be used to monitor temperature of in vitro experiments, and it is also able to be taken to explore regulation of pUb function by temperature. Furthermore, the ¹⁹F-labeled pUb thermosensor could be developed to detect intracellular temperature in-cell NMR assays.

Key words: ubiquitin, phosphorylation, nuclear magnetic resonance (NMR), thermosensor

中图分类号:

O482.53

张志武,杨菊,聂泽锋,叶尚祥,董旭,唐淳. 基于¹⁹F化学标记磷酸化泛素的温度传感器开发[J]. 波谱学杂志, 2021, 38(2): 173-181.

Zhi-wu ZHANG,Ju YANG,Ze-feng NIE,Shang-xiang YE,Xu DONG,Chun TANG. Development of a Temperature Senor Based on ¹⁹F-labeled Phosphorylated Ubiquitin[J]. Chinese Journal of Magnetic Resonance, 2021, 38(2): 173-181.

图/表 6

图1

图2

图3

图4

图5

图6

参考文献 28

1	BLOCK B A . Thermogenesis in muscle[J]. Annu Rev Physiol, 1994, 56 (1): 535- 577. doi: 10.1146/annurev.ph.56.030194.002535
2	DUHR S , BRAUN D . Why molecules move along a temperature gradient[J]. Proc Natl Acad Sci U S A, 2006, 103 (52): 19678- 19682. doi: 10.1073/pnas.0603873103
3	YANG J M , YANG H , LIN L W . Quantum dot nano thermometers reveal heterogeneous local thermogenesis in living cells[J]. ACS Nano, 2011, 5 (6): 5067- 5071. doi: 10.1021/nn201142f
4	NICHOLLS D G , LOCKE R M . Thermogenic mechanisms in brown fat[J]. Physiol Rev, 1984, 64 (1): 1- 64. doi: 10.1152/physrev.1984.64.1.1
5	SILVA J E . Thermogenic mechanisms and their hormonal regulation[J]. Physiol Rev, 2006, 86 (2): 435- 464. doi: 10.1152/physrev.00009.2005
6	BAL N C , MAURYA S K , SOPARIWALA D H , et al. Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals[J]. Nat Med, 2012, 18 (10): 1575- 1579. doi: 10.1038/nm.2897
7	GARAMI A , STEINER A A , ROMANOVSKY A A . Fever and hypothermia in systemic inflammation[J]. Handb Clin Neurol, 2018, 157, 565- 597.
8	BLOMQVIST A , ENGBLOM D . Neural mechanisms of inflammation-induced fever[J]. Neuroscientist, 2018, 24 (4): 381- 399. doi: 10.1177/1073858418760481
9	CHRETIEN D , BÉNIT P , HA H H , et al. Mitochondria are physiologically maintained at close to 50℃[J]. PLoS Biol, 2018, 16 (1): e2003992. doi: 10.1371/journal.pbio.2003992
10	ANDREWS Z B , DIANO S , HORVATH T L . Mitochondrial uncoupling proteins in the CNS: in support of function and survival[J]. Nat Rev Neurosci, 2005, 6 (11): 829- 840. doi: 10.1038/nrn1767
11	ARAI S , SUZUKI M , PARK S J , et al. Mitochondria-targeted fluorescent thermometer monitors intracellular temperature gradient[J]. Chem Commun, 2015, 51 (38): 8044- 8047. doi: 10.1039/C5CC01088H
12	OKABE K , SAKAGUCHI R , KIYONAKA S , et al. Intracellular thermometry with fluorescent sensors for thermal biology[J]. Pflugers Arch, 2018, 470 (5): 717- 731. doi: 10.1007/s00424-018-2113-4
13	NAKANO M , ARAI Y , IPPEI KOTERA I , et al. Genetically encoded ratiometric fluorescent thermometer with wide range and rapid response[J]. PLoS ONE, 2017, 12 (2): e0172344. doi: 10.1371/journal.pone.0172344
14	CHOWDHURY S , MARIS C , ALLAIN F H T , et al. Molecular basis for temperature sensing by an RNA thermometer[J]. EMBO J, 2006, 25 (11): 2487- 2497. doi: 10.1038/sj.emboj.7601128
15	VIJAY-KUMAR S , BUGG C E , COOK W J . Structure of ubiquitin refined at 1.8Å[J]. J Mol Biol, 1987, 194 (3): 531- 544. doi: 10.1016/0022-2836(87)90679-6
16	KOMANDER D , RAPE M . The ubiquitin code[J]. Annu Rev Biochem, 2012, 81, 203- 229. doi: 10.1146/annurev-biochem-060310-170328
17	TANG C , ZHANG W P . How phosphorylation by PINK1 remodels the ubiquitin system: a perspective from structure and dynamics[J]. Biochemistry, 2020, 59 (1): 26- 33. doi: 10.1021/acs.biochem.9b00715
18	KANE L A , LAZAROU M , AI F , et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity[J]. J Cell Biol, 2014, 205 (2): 143- 153. doi: 10.1083/jcb.201402104
19	YE S X , GONG Z , YANG J , et al. Ubiquitin is double-phosphorylated by PINK1 for enhanced pH-sensitivity of conformational switch[J]. Protein Cell, 2019, 10 (12): 908- 913. doi: 10.1007/s13238-019-0644-x
20	KOYANO F , OKATSU K , KOSAKO H , et al. Ubiquitin is phosphorylated by PINK1 to activate parkin[J]. Nature, 2014, 510 (7503): 162- 166. doi: 10.1038/nature13392
21	DONG X , GONG Z , LU Y B , et al. Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch[J]. Proc Natl Acad Sci U S A, 2017, 114 (26): 6770- 6775.
22	WAUER T , SWATEK K N , WAGSTAFF J L , et al. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis[J]. EMBO J, 2015, 34 (3): 307- 325. doi: 10.15252/embj.201489847
23	AN Y F , CHEN L M , SUN S H , et al. QuikChange shuffling: a convenient and robust method for site-directed mutagenesis and random recombination of homologous genes[J]. N Biotechnol, 2011, 28 (4): 320- 325. doi: 10.1016/j.nbt.2011.03.001
24	CHEN H F , VIEL S M , ZIARELLI F H , et al. ¹⁹F NMR: a valuable tool for studying biological events[J]. Chem Soc Rev, 2013, 42 (20): 7971- 7982. doi: 10.1039/c3cs60129c
25	KITEVSKI-LEBLANC J L , PROSSER R S . Current applications of ¹⁹F NMR to studies of protein structure and dynamics[J]. Prog Nucl Magn Reson Spectrosc, 2012, 62, 1- 33. doi: 10.1016/j.pnmrs.2011.06.003
26	SHEKHAWAT S S , PHAM G H , PRABAKARAN J , et al. Simultaneous detection of distinct ubiquitin chain topologies by ¹⁹F NMR[J]. ACS Chem Biol, 2014, 9 (10): 2229- 2236. doi: 10.1021/cb500589c
27	DANIELSON M A , FALKE J J . Use of ¹⁹F NMR to probe protein structure and conformational changes[J]. Annu Rev Biophys Biomol Struct, 1996, 25, 163- 195. doi: 10.1146/annurev.bb.25.060196.001115
28	GOOD N E , DOUGLAS WINGET G , WINTER W , et al. Hydrogen ion buffers for biological research[J]. Biochemistry, 1966, 5 (2): 467. doi: 10.1021/bi00866a011

[1]	王崇武,黄曦,石磊,陈世桢,周欣. 组织蛋白酶B响应的超极化¹²⁹Xe MRI探针对肺癌细胞的超灵敏探测[J]. 波谱学杂志, 2021, 38(3): 336-344.
[2]	牛星星,白志杰,杨翼,高杨文,王雪璐,姚叶锋. 原位低场核磁共振弛豫法定量监测光催化Cr(Ⅵ)还原反应[J]. 波谱学杂志, 2021, 38(3): 403-413.
[3]	谢金华,王乐,刘盼,苏稀琪,MINGLi-June. 盐酸四环素中可交换氢和氢键的核磁共振波谱研究[J]. 波谱学杂志, 2021, 38(3): 313-322.
[4]	吴嘉敏,贺玉成,徐征,朱延河,姜文正. 用于土壤水分测量的磁共振射频线圈宽频匹配方法[J]. 波谱学杂志, 2021, 38(3): 414-423.
[5]	王子豪,徐赫,汪涛,杨善中,丁运生,魏海兵. 外型和内型C-2位单取代降冰片烯衍生物的核磁共振波谱研究[J]. 波谱学杂志, 2021, 38(3): 323-335.
[6]	黄兴,张子立,胡新宁,牛飞飞,孙万硕,孔祥东,戴银明. 核磁共振磁体超导接头工艺研究进展[J]. 波谱学杂志, 2021, 38(3): 424-432.
[7]	张书怀,马慧,郭朝晖,马敏珺,乔岩,王英雄. 正丁醇/丙酸与腐殖酸相互作用的NMR研究[J]. 波谱学杂志, 2021, 38(3): 301-312.
[8]	赵心怡,韩冬,罗红军,沈文斌,杨功俊. 德拉沙星葡甲胺波谱学数据解析[J]. 波谱学杂志, 2021, 38(2): 268-276.
[9]	陈晓雯,黄碧玲,黄少华,赵玉芬. Bacillus subtilis转录因子CtsR蛋白中clpC操纵子结合区域的NMR研究[J]. 波谱学杂志, 2021, 38(2): 155-163.
[10]	随松,高国梁,王雪璐,魏达秀,姚叶锋. 固-液-气三相环境下非均相苯加氢反应的原位核磁共振研究[J]. 波谱学杂志, 2021, 38(2): 194-203.
[11]	李毅,辛家祥,王嘉琛,魏达秀,姚叶锋. 三种脉冲序列制备核自旋单重态效率的比较[J]. 波谱学杂志, 2021, 38(2): 227-238.
[12]	余锦波,张偲,张则婷,徐国华,李从刚. Alpha-突触核蛋白与完整线粒体相互作用的NMR研究[J]. 波谱学杂志, 2021, 38(2): 164-172.
[13]	窦梦雨,赵奇,侯相林,刘雷,唐明兴,王英雄. 蒽加氢产物的结构指认和定量核磁共振分析[J]. 波谱学杂志, 2021, 38(2): 239-248.
[14]	张伟,吴意明,崔维平,肖亮. 稠油储层核磁共振孔隙度校正方法[J]. 波谱学杂志, 2021, 38(2): 204-214.
[15]	孟昆,王胜建,薛宗安,侯瑞卿,肖亮. 利用核磁共振资料定量评价页岩孔隙结构[J]. 波谱学杂志, 2021, 38(2): 215-226.

基于¹⁹F化学标记磷酸化泛素的温度传感器开发

Development of a Temperature Senor Based on ¹⁹F-labeled Phosphorylated Ubiquitin

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价