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

doi:10.11938/cjmr20182625

Abstract

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

CLC Number:

O482.53

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.

References

[1] DOIG A J, DERREUMAUX P. Inhibition of protein aggregation and amyloid formation by small molecules[J]. Curr Opin Struct Biol, 2015, 30:50-56.
[2] YANG-HARTWICH Y, SOTERAS M G, LIN Z P, et al. p53 protein aggregation promotes platinum resistance in ovarian cancer[J]. Oncogene, 2015, 34(27):3605-3616.
[3] CHEN Y H, ZHANG Z T, BAI J, et al. Inhibition mechanisms of protein disulfide isomerase on a-synuclein Fibril aggregation[J].Chinese J Magn Reson, 2017, 34(2):131-136. 陈艳华, 张则婷, 白佳, 等. PDI抑制α-synuclein纤维化聚集作用机制研究[J]. 波谱学杂志, 2017, 34(2):131-136.
[4] JOHNSON JR C S. Diffusion ordered nuclear magnetic resonance spectroscopy:principles and applications[J]. Prog NMR Spect, 1999, 34:203-256.
[5] KE E F, CHEN Z, YE J L, et al. 2D diffusion-ordered nuclear magnetic resonance spectroscopy for structure analysis of mixtures[J]. Journal of Xiamen University-Natural Science, 1999, 38(3):366-370. 柯恩烽, 陈忠, 叶剑良, 等. 混合物溶液结构的二维扩散排序核磁共振谱[J]. 厦门大学学报(自然科学版), 1999, 38(3):366-370.
[6] DEHNER A, KESSLER H. Diffusion NMR spectroscopy:folding and aggregation of domains in p53[J]. Chem Bio Chem, 2005, 6(9):1550-1565.
[7] MACCHIONI A, CIANCALEONI G, ZUCCACCIA C, et al. Determining accurate molecular sizes in solution through NMR diffusion spectroscopy[J]. Chem Soc Rev, 2008, 37(3):479-489.
[8] FLOQUET S, BRUN S, LEMONNIER J F, et al. Molecular weights of cyclic and hollow clusters measured by DOSY NMR spectroscopy[J]. J Am Chem Soc, 2009, 131(47):17254-17259.
[9] MALLOL R, RODRÍGUEZ M A, HERAS M, et al. Particle size measurement of lipoprotein fractions using diffusion-ordered NMR spectroscopy[J]. Anal Bioanal Chem., 2012, 402(7):2407-2415.
[10] ZHANG F, YU X Y, CHEN Z, et al. Characterizations of some N-substituted-salicylhydrazide in mixtures by NMR diffusion ordered spectroscopy[J]. Chinese J Struct Chem, 2003, 22(3):287-292.
[11] MANSFIELD S L, JAYAWICKRAMA D A, TIMMONS J S, et al. Measurement of peptide aggregation with pulsed-field gradient nuclear magnetic resonance spectroscopy[J]. Biochim Biophys Acta, 1998, 1382(2):257-265.
[12] YAO S, HOWLETT G J, NORDON R S. Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements[J]. J Biomol NMR, 2000, 16(2):109-119.
[13] CHEN C G, ZHANG M M, LIU Y P. Study on the Interaction between β-Cyclodextrin and naproxen by two-dimensional diffusion ordered NMR Spectroscopy[J]. J Instrum Anal, 2011, 30(9):959-963. 陈昌国, 张明明, 刘渝萍. 二维扩散排序核磁共振谱法研究β-环糊精/萘普生的相互作用[J]. 分析测试学报, 2011, 30(9):959-963.
[14] PRICE W S, NARA M, ARATA Y. A pulsed field gradient NMR study of the aggregation and hydration of parvalbumin[J]. Biophys Chem, 1997, 65(2, 3):179-187.
[15] LIN M, LARIVE C K. Detection of insulin aggregates with pulsed-field gradient nuclear magnetic resonance spectroscopy[J]. Anal Biochem, 1995, 229(2):214-220.
[16] AUGEx S, SCHMIT P, CRUTCHFIELD C A, et al. NMR measure of translational diffusion and fractal dimension. Application to molecular mass measurement[J]. J Phys Chem B, 2009, 113(7):1914-1918.
[17] WANG L M, QIU R C, HUANG S H. Quantitative analysis of active ingredients in compound acetylsalicylic acid tablets by DOSY[J]. Chinese J Magn Reson, 2016, 33(3):415-421. 王丽敏, 仇汝臣, 黄少华. 复方乙酰水杨酸片中有效成分的DOSY技术分析[J]. 波谱学杂志, 2016, 33(3):415-421.
[18] ZHONG N, ZHANG S N, ZOU P, et al. Without its N-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its C-terminal domain[J]. J Virol, 2008, 82(9):4227-4234.