Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2
Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2
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DOI：
10.1007/bf00197813
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发表时间：
1995-11
影响因子：
2.7
通讯作者：
A. Dingley;J. Mackay;B. Chapman;M. Morris;P. Kuchel;B. Hambly;G. King
中科院分区：
文献类型：
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作者：
A. Dingley;J. Mackay;B. Chapman;M. Morris;P. Kuchel;B. Hambly;G. King
在结构研究所需的毫摩尔浓度下，钙结合蛋白肌球蛋白轻链2（MLC 2）的NMR光谱显示共振线宽指示广泛的自缔合。脉冲场梯度（PFG）NMR光谱用于检查MLC 2聚集是否可以通过两性离子胆汁盐衍生物3-[（3-胆酰胺丙基）二甲基铵基]-1-丙磺酸盐（CHAPS）来防止。PFG NMR测量表明，CHAPS是能够防止MLC 2自缔合，但只有在浓度远高于临界胶束浓度的107.5 mM。CHAPS是最有效的浓度为22.5 mM，其中MLC 2的表观分子量对应于蛋白质单体加上7个分子的结合洗涤剂。MLC 2的2D 15 N-1H HSQC光谱的分辨率和灵敏度通过加入25 mM CHAPS而显著提高，这与加入去污剂后聚集减少一致。MLC 2的平均酰胺氮T2值从不存在CHAPS的0.30 ms增加到存在25 mM CHAPS的0.56 ms。本研究的结果使我们提出，PFG NMR光谱可以作为一个简单的替代传统的技术，如分析ultracenthegation检查生物大分子的自缔合。
At the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of ∼7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 correponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D15N-1H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T2value for MLC2 increased from ∼30 ms in the absence of CHAPS to ∼56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules.