硫氧还蛋白重组体的魔角旋转核磁共振光谱学

Magic angle spinning NMR spectroscopy of thioredoxin reassemblies.

作者信息

Yang Jun, Paramasivam Sivakumar, Marulanda Dabeiba, Cataldi Marcela, Tasayco Maria Luisa, Polenova Tatyana

机构信息

Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.

出版信息

Magn Reson Chem. 2007 Dec;45 Suppl 1:S73-83. doi: 10.1002/mrc.2092.

DOI:10.1002/mrc.2092

PMID:18157811

Abstract

Differentially isotopically enriched 1-73((13)C,(15)N)/74-108((15)N) and 1-73((15)N)/74-108((13)C,(15)N) Escherichia coli thioredoxin reassemblies prepared by fragment complementation were investigated by high-resolution magic angle spinning solid-state NMR spectroscopy. Nearly complete resonance assignments, secondary and tertiary structure analysis are reported for 1-73((13)C,(15)N)/74-108((15)N) reassembled thioredoxin. Temperature dependence of the dipolar-assisted rotational resonance (DARR) spectra reveals the residues undergoing intermediate timescale motions at temperatures below - 15 degrees C. Analysis of the DARR intensity buildups as a function of mixing time in these reassemblies indicates that at long mixing times medium- and long-range cross-peaks do not experience dipolar truncation, suggesting that isotopic dilution is not required for gaining nontrivial distance restraints for structure calculations.

摘要

通过片段互补制备的差异同位素富集的1-73（（13）C，（15）N）/ 74-108（（15）N）和1-73（（15）N）/ 74-108（（13）C，（15）N）大肠杆菌硫氧还蛋白重组体，采用高分辨率魔角旋转固态核磁共振光谱进行了研究。报道了1-73（（13）C，（15）N）/ 74-108（（15）N）重组硫氧还蛋白几乎完整的共振归属、二级和三级结构分析。偶极辅助旋转共振（DARR）光谱的温度依赖性揭示了在低于-15℃的温度下经历中间时间尺度运动的残基。对这些重组体中作为混合时间函数的DARR强度积累的分析表明，在长混合时间下，中程和远程交叉峰不会经历偶极截断，这表明在进行结构计算时获得重要的距离限制不需要同位素稀释。