扩散序谱(DOSY)实验测定缓冲体系中蛋白质表观分子量

doi:10.11938/cjmr20182625

波谱学杂志 ›› 2018, Vol. 35 ›› Issue (3): 280-286.doi: 10.11938/cjmr20182625

扩散序谱(DOSY)实验测定缓冲体系中蛋白质表观分子量

李红卫^1,2, 袁志良^1,3, 夏斌^1,2,3

1. 北京大学北京核磁共振中心, 北京 100871;
2. 北京大学化学与分子工程学院, 北京 100871;
3. 北京大学生命科学学院, 北京 100871

收稿日期:2018-03-27 出版日期:2018-09-05 发布日期:2018-08-28
通讯作者: 李红卫,Tel:010-62756056,E-mail:lihongwei@pku.edu.cn E-mail:lihongwei@pku.edu.cn
基金资助:
国家重点研发计划资助项目（2016YFA0501203）；国家自然科学基金青年科学基金资助项目（21705005）.

Determination of Apparent Protein Molecular Weight in Solution by Diffusion Ordered NMR Spectroscopy

LI Hong-wei^1,2, YUAN Zhi-liang^1,3, XIA Bin^1,2,3

1. Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China;
2. College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China;
3. College of Life Sciences, Peking University, Beijing 100871, China

Received:2018-03-27 Online:2018-09-05 Published:2018-08-28

1. 补充材料.pdf(694KB)

摘要/Abstract

摘要： 液体核磁共振扩散序谱（DOSY）可以通过测定溶质分子的自扩散系数（D_t）来研究该分子在溶液中的表观分子量（M）.D_t与测试体系和分子本身性质相关，蛋白质体系较为复杂，从而增加了蛋白质自扩散系数（D_t-protein）测定的难度.本文以3-（三甲基硅基）丙磺酸钠（DSS）为内标，以蛋白质分子与DSS自扩散系数的比值（D_r）来表征蛋白质分子在溶液中的表观分子量（M_protein），该方法降低了缓冲体系对M_protein的影响，使得M_protein主要由分子本身的性质决定.在此基础上，测定了不同分子量蛋白质分子相对于DSS的D_r，拟合得到了D_r与M_protein的相关关系：lgM_protein =-2.6488 lgD_r-0.7863，相关系数（R²）为0.997.最后测定了通过大肠杆菌表达纯化得到的SARS冠状病毒主蛋白酶C端结构域（M^pro-C）分子相对于DSS的D_r，并计算出与文献结果一致的M_protein，进一步验证了拟合公式的准确性和实用性.

关键词: 液体核磁共振(liquid-state NMR), 表观分子量, 扩散序谱(DOSY), 缓冲体系, 脉冲梯度场

Abstract: Diffusion ordered NMR spectroscopy (DOSY) can be used to determine the apparent molecular weight of proteins (M_protein) by measuring self-diffusion coefficient (D_t-protein). However, D_t-protein is also affected by the property of buffer solution, making the measurements complicated. In this work, 3-(trimethylsilyl) propane sodium sulfonate (DSS) was used as the internal reference molecule and the diffusion coefficient ratio between the target protein and DSS (D_r) was used to characterize the protein in solution. This method reduced the effects of buffer system on M_protein determination, yielding more accurate M_protein measurement. The D_r values of reference proteins with different molecular weight were determined. A correlation curve between D_r and M_protein was established (lgM_protein=-2.6488 lgD_r-0.7863, R²=0.997). Finally, D_r of the C-terminal domain of SARS-CoV main protease (M^pro-C) was determined and to yield its molecular weight. Accuracy and practicability of the fitting curve were demonstrated.

Key words: liquid-state NMR, apparent molecular weight, diffusion ordered NMR spectroscopy (DOSY), buffer solution, pulse gradient

中图分类号:

O482.53

李红卫, 袁志良, 夏斌. 扩散序谱(DOSY)实验测定缓冲体系中蛋白质表观分子量[J]. 波谱学杂志, 2018, 35(3): 280-286.

LI Hong-wei, YUAN Zhi-liang, XIA Bin. Determination of Apparent Protein Molecular Weight in Solution by Diffusion Ordered NMR Spectroscopy[J]. Chinese Journal of Magnetic Resonance, 2018, 35(3): 280-286.

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扩散序谱(DOSY)实验测定缓冲体系中蛋白质表观分子量

Determination of Apparent Protein Molecular Weight in Solution by Diffusion Ordered NMR Spectroscopy

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