非天然氨基酸在蛋白质动态特性核磁共振研究中的应用

doi:10.11938/cjmr20212931

摘要/Abstract

摘要：

核磁共振（NMR）是蛋白质结构解析的主要方法之一.除可获得蛋白质的高分辨结构外，NMR还可用于观测最接近生理条件的蛋白质动态构象，获得蛋白质行使生物学功能的详细机制.非天然氨基酸定点标记方法可显著减少大分子量蛋白质的信号数目，降低数据采集和分析难度，已广泛运用于蛋白质结构和功能研究.本文介绍了常用的在蛋白质中引入非天然氨基酸的实验方法，包括蛋白质化学合成法、蛋白质化学修饰法、氟代芳香族氨基酸插入和基因密码子编辑的位点特异性插入等方法，并介绍了部分应用非天然氨基酸结合NMR研究大分子量蛋白的成功案例.此外，此篇综述讨论了目前非天然氨基酸标记在蛋白质研究中的局限性及发展方向.

关键词: 液体核磁共振, 固体核磁共振, 非天然氨基酸, 大分子量蛋白质

Abstract:

Nuclear magnetic resonance (NMR) is a major method used to study protein structure at atomic resolution. Besides presenting the high-resolution structure, NMR facilitates studies on protein dynamics near physiological conditions that are intimately related to the biological mechanism of the proteins. Unnatural amino acids (UAA) labeling could significantly reduce the complexity of protein NMR spectra. In this review, we briefly summarize the widely used UAA labeling strategies for proteins, including chemical peptide synthesis, residue-specific peptide modification, ¹⁹F labeled aromatic amino acids incorporation and genetic code expansion based UAA incorporation. The recent applicants of UAA in characterizing protein structure and dynamics, the limitations and prospects of UAA are also highlighted.

Key words: solution NMR, solid-state NMR, unnatural amino acid, large size protein

中图分类号:

O482.53

史朝为,石攀,田长麟. 非天然氨基酸在蛋白质动态特性核磁共振研究中的应用[J]. 波谱学杂志, 2021, 38(4): 523-532.

Chao-wei SHI,Pan SHI,Chang-lin TIAN. NMR Studies of Large Protein Dynamics Using Unnatural Amino Acids[J]. Chinese Journal of Magnetic Resonance, 2021, 38(4): 523-532.

图/表 4

参考文献 44

1	HENZLER-WILDMAN K , KERN D . Dynamic personalities of proteins[J]. Nature, 2007, 450 (7172): 964- 972. doi: 10.1038/nature06522
2	MERRIFIELD R B . Solid phase peptide synthesis. I. the synthesis of a tetrapeptide[J]. J Am Chem Soc, 1963, 85 (14): 2149- 2154. doi: 10.1021/ja00897a025
3	YU H M , CHEN S T , WANG K T . Enhanced coupling efficiency in solid-phase peptide synthesis by microwave irradiation[J]. J Org Chem, 1992, 57 (18): 4781- 4784. doi: 10.1021/jo00044a001
4	GORDON C P . The renascence of continuous-flow peptide synthesis-an abridged account of solid and solution-based approaches[J]. Org Biomol Chem, 2018, 16 (2): 180- 196. doi: 10.1039/C7OB02759A
5	FANG G M , LI Y M , SHEN F , et al. Protein chemical synthesis by ligation of peptide hydrazides[J]. Angew Chem Int Ed Engl, 2011, 50 (33): 7645- 7649. doi: 10.1002/anie.201100996
6	LIU J J , HORST R , KATRITCH V , et al. Biased signaling pathways in beta2-adrenergic receptor characterized by ¹⁹F-NMR[J]. Science, 2012, 335 (6072): 1106- 1110. doi: 10.1126/science.1215802
7	LUCHETTE P A , PROSSER R S , SANDERS C R . Oxygen as a paramagnetic probe of membrane protein structure by cysteine mutagenesis and ¹⁹F NMR spectroscopy[J]. J Am Chem Soc, 2002, 124 (8): 1778- 1781. doi: 10.1021/ja016748e
8	SALWICZEK M , SAMSONOV S , VAGT T , et al. Position-dependent effects of fluorinated amino acids on the hydrophobic core formation of a heterodimeric coiled coil[J]. Chemistry, 2009, 15 (31): 7628- 7636. doi: 10.1002/chem.200802136
9	HATTORI Y , HEIDENREICH D , ONO Y , et al. Protein ¹⁹F-labeling using transglutaminase for the NMR study of intermolecular interactions[J]. J Biomol NMR, 2017, 68 (4): 271- 279. doi: 10.1007/s10858-017-0125-6
10	LARDA S T , BOKOCH M P , EVANICS F , et al. Lysine methylation strategies for characterizing protein conformations by NMR[J]. J Biomol NMR, 2012, 54 (2): 199- 209. doi: 10.1007/s10858-012-9664-z
11	LUCK L A , FALKE J J . ¹⁹F NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change[J]. Biochemistry, 1991, 30 (17): 4248- 4256. doi: 10.1021/bi00231a021
12	LI H , FRIEDEN C . NMR studies of 4-¹⁹F-phenylalanine-labeled intestinal fatty acid binding protein: evidence for conformational heterogeneity in the native state[J]. Biochemistry, 2005, 44 (7): 2369- 2377. doi: 10.1021/bi047600l
13	ROPSON I J , BOYER J A , DALESSIO P M . A residual structure in unfolded intestinal fatty acid binding protein consists of amino acids that are neighbors in the native state[J]. Biochemistry, 2006, 45 (8): 2608- 3617. doi: 10.1021/bi052091o
14	ANDERLUH G , RAZPOTNIK A , PODLESEK Z , et al. Interaction of the eukaryotic pore-forming cytolysin equinatoxin Ⅱ with model membranes: ¹⁹F NMR studies[J]. J Mol Biol, 2005, 347 (1): 27- 39. doi: 10.1016/j.jmb.2004.12.058
15	BANN J G , PINKNER J , HULTGREN S J , et al. Real-time and equilibrium ¹⁹F-NMR studies reveal the role of domain-domain interactions in the folding of the chaperone PapD[J]. Proc Natl Acad Sci U S A, 2002, 99 (2): 709- 714. doi: 10.1073/pnas.022649599
16	LEE H W , SOHN J H , YEH B I , et al. ¹⁹F NMR investigation of F(1)-ATPase of Escherichia coli using fluorotryptophan labeling[J]. J Biochem, 2000, 127 (6): 1053- 1056. doi: 10.1093/oxfordjournals.jbchem.a022697
17	ZHANG Y , WANG L , SCHULTZ P G , et al. Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine[J]. Protein Sci, 2005, 14 (5): 1340- 1349. doi: 10.1110/ps.041239305
18	YOUNG T S , AHMAD I , YIN J A , et al. An enhanced system for unnatural amino acid mutagenesis in E. coli[J]. J Mol Biol, 2010, 395 (2): 361- 374. doi: 10.1016/j.jmb.2009.10.030
19	HORST R , LIU J J , STEVENS R C , et al. beta(2)-adrenergic receptor activation by agonists studied with ¹⁹F NMR spectroscopy[J]. Angew Chem Int Ed Engl, 2013, 52 (41): 10762- 10765. doi: 10.1002/anie.201305286
20	WANG H X , HU W H , LIU D S , et al. Design and preparation of the class B G protein-coupled receptors GLP-1R and GCGR for ¹⁹F-NMR studies in solution[J]. FEBS J, 2021, 288 (13): 4053- 4063. doi: 10.1111/febs.15686
21	MA X Y , HU Y F , BATEBI H , et al. Analysis of beta2AR-Gs and beta2AR-Gi complex formation by NMR spectroscopy[J]. Proc Natl Acad Sci U S A, 2020, 117 (37): 23096- 23105. doi: 10.1073/pnas.2009786117
22	STERNBERG U , KLIPFEL M , GRAGE S L , et al. Calculation of fluorine chemical shift tensors for the interpretation of oriented ¹⁹F-NMR spectra of gramicidin A in membranes[J]. Phys Chem Chem Phys, 2009, 11 (32): 7048- 7060. doi: 10.1039/b908236k
23	KHAN F , KUPROV I , CRAGGS T D , et al. ¹⁹F NMR studies of the native and denatured states of green fluorescent protein[J]. J Am Chem Soc, 2006, 128 (33): 10729- 10737. doi: 10.1021/ja060618u
24	EVANICS F , BEZSONOVA I , MARSH J , et al. Tryptophan solvent exposure in folded and unfolded states of an SH3 domain by ¹⁹F and ¹H NMR[J]. Biochemistry, 2006, 45 (47): 14120- 14128. doi: 10.1021/bi061389r
25	YE Y S , LIU X L , XU G H , et al. Direct observation of Ca²⁺-induced calmodulin conformational transitions in intact Xenopus laevis oocytes by ¹⁹F NMR spectroscopy[J]. Angew Chem Int Ed Engl, 2015, 54 (18): 5328- 5330. doi: 10.1002/anie.201500261
26	SPEER S L, ZHENG W, JIANG X, et al. The intracellular environment affects protein-protein interactions[J]. Proc Natl Acad Sci U S A, 2021, 118(11): e2019918118.
27	YE Y S , WU Q , ZHENG W W , et al. Quantification of size effect on protein rotational mobility in cells by ¹⁹F NMR spectroscopy[J]. Anal Bioanal Chem, 2018, 410 (3): 869- 874. doi: 10.1007/s00216-017-0745-4
28	SHI P , XI Z Y , WANG H , et al. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded ¹⁵N/¹⁹F-labeled unnatural amino acid[J]. Biochem Biophys Res Commun, 2010, 402 (3): 461- 466. doi: 10.1016/j.bbrc.2010.10.046
29	SHI P , WANG H , XI Z Y , et al. Site-specific ¹⁹F NMR chemical shift and side chain relaxation analysis of a membrane protein labeled with an unnatural amino acid[J]. Protein Sci, 2011, 20 (1): 224- 228. doi: 10.1002/pro.545
30	SHI P , LI D , CHEN H , et al. Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and ¹⁹F nuclear magnetic resonance[J]. Biochem Biophys Res Commun, 2011, 414 (2): 379- 383. doi: 10.1016/j.bbrc.2011.09.082
31	SHI P , LI D , LAI C H , et al. Intracellular segment between transmembrane helices S0 and S1 of BK channel alpha subunit contains two amphipathic helices connected by a flexible loop[J]. Biochem Biophys Res Commun, 2013, 437 (3): 408- 412. doi: 10.1016/j.bbrc.2013.06.091
32	SHI P , LI D , CHEN H W , et al. In situ ¹⁹F NMR studies of an E. coli membrane protein[J]. Protein Sci, 2012, 21 (4): 596- 600. doi: 10.1002/pro.2040
33	GUO X , WANG L , LI J , et al. Structural insight into autoinhibition and histone H3-induced activation of DNMT3A[J]. Nature, 2015, 517 (7536): 640- 644. doi: 10.1038/nature13899
34	LI Y J , HAN J M , ZHANG Y B , et al. Structural basis for activity regulation of MLL family methyltransferases[J]. Nature, 2016, 530 (7591): 447- 452. doi: 10.1038/nature16952
35	ZHU K , SHAN Z , CHEN X , et al. Allosteric auto-inhibition and activation of the Nedd4 family E3 ligase Itch[J]. EMBO Rep, 2017, 18 (9): 1618- 1630. doi: 10.15252/embr.201744454
36	YANG F , YU X , LIU C , et al. Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and ¹⁹F NMR[J]. Nat Commun, 2015, 6, 8202. doi: 10.1038/ncomms9202
37	LIU Q , HE Q T , LYU X , et al. DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl ¹H-NMR probe[J]. Nat Commun, 2020, 11 (1): 4857. doi: 10.1038/s41467-020-18433-5
38	HE Q T , XIAO P , HUANG S M , et al. Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2[J]. Nat Commun, 2021, 12 (1): 2396. doi: 10.1038/s41467-021-22731-x
39	LI D , LI J , ZHUANG Y L , et al. Nano-size uni-lamellar lipodisq improved in situ auto-phosphorylation analysis of E. coli tyrosine kinase using ¹⁹F nuclear magnetic resonance[J]. Protein Cell, 2015, 6 (3): 229- 233. doi: 10.1007/s13238-014-0129-x
40	LI D , ZHANG Y N , HE Y , et al. Protein-protein interaction analysis in crude bacterial lysates using combinational method of ¹⁹F site-specific incorporation and ¹⁹F NMR[J]. Protein Cell, 2017, 8 (2): 149- 154. doi: 10.1007/s13238-016-0336-8
41	POLENOVA T , GUPTA R , GOLDBOURT A . Magic angle spinning NMR spectroscopy: a versatile technique for structural and dynamic analysis of solid-phase systems[J]. Anal Chem, 2015, 87 (11): 5458- 5469. doi: 10.1021/ac504288u
42	ROOS M , WANG T , SHCHERBAKOV A A , et al. Fast magic-angle-spinning ¹⁹F spin exchange NMR for determining nanometer ¹⁹F-¹⁹F distances in proteins and pharmaceutical compounds[J]. J Phys Chem B, 2018, 122 (11): 2900- 2911. doi: 10.1021/acs.jpcb.8b00310
43	SHCHERBAKOV A A , HONG M . Rapid measurement of long-range distances in proteins by multidimensional ¹³C-¹⁹F REDOR NMR under fast magic-angle spinning[J]. J Biomol NMR, 2018, 71 (1): 31- 43. doi: 10.1007/s10858-018-0187-0
44	XUE K , SARKAR R , MOTZ C , et al. Limits of resolution and sensitivity of proton detected MAS solid-state NMR experiments at 111 kHz in deuterated and protonated proteins[J]. Sci Rep, 2017, 7 (1): 7444. doi: 10.1038/s41598-017-07253-1

[1]	陈翰迪,孔海宇,赵侦超,张维萍. 固体核磁共振结合密度泛函理论计算研究SSZ-39分子筛的钠离子落位与铝分布[J]. 波谱学杂志, 2021, 38(4): 543-551.
[2]	杨文杰,黄骏. 基于固体核磁共振技术的固体酸结构、酸性及活性分析[J]. 波谱学杂志, 2021, 38(4): 460-473.
[3]	夏锡锋,张文静,林芝晔,柯晓康,温玉洁,王芳,陈俊超,彭路明. 氧化物纳米材料表面结构与性质的固体核磁共振波谱研究[J]. 波谱学杂志, 2021, 38(4): 533-542.
[4]	王永祥,王强,徐君,夏清华,邓风. 六氟硅酸铵后处理对H-ZSM-5分子筛酸性影响的固体NMR研究[J]. 波谱学杂志, 2021, 38(4): 514-522.
[5]	王子春,黄骏,姜怡娇. 五配位铝强化硅铝固体酸的固体核磁共振研究[J]. 波谱学杂志, 2021, 38(4): 552-570.
[6]	肖瑶,夏长久,易先锋,刘凤庆,刘尚斌,郑安民. 固体核磁共振技术在锡硅分子筛表征中的应用[J]. 波谱学杂志, 2021, 38(4): 571-584.
[7]	高树树,徐舒涛,魏迎旭,刘中民. 固体核磁共振技术在甲醇制烯烃反应中的应用[J]. 波谱学杂志, 2021, 38(4): 433-447.
[8]	陈欣,付颖懿,岳斌,贺鹤勇. 固体核磁共振技术研究金属氧化物类固体酸催化剂的酸碱性[J]. 波谱学杂志, 2021, 38(4): 491-502.
[9]	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.
[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]	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.
[12]	李玉江, 赵伟, 郭晓河, 陶乐, 张祥, 张海艳, 赵天增. 盐酸马尼地平的核磁共振数据解析[J]. 波谱学杂志, 2021, 38(1): 110-117.
[13]	王睿迪, 徐贝贝, 宋艳红, 王雪璐, 姚叶锋. 原位核磁共振技术研究光催化甲醇重整过程中甲醇与水的相互作用[J]. 波谱学杂志, 2021, 38(1): 43-57.
[14]	黄珊珊, 姚叶锋, 李鹏, 何培忠. 液体核磁共振 HSQC 实验的量子化学计算与模拟[J]. 波谱学杂志, 2021, 38(1): 32-42.
[15]	周中高, 谢倩, 元洋洋, 李静, 路东亮, 陈正旺. 吡喃葡糖基氮杂环卡宾-钯(II)-吡啶配合物的NMR研究[J]. 波谱学杂志, 2020, 37(4): 505-514.