Marulanda Dabeiba, Tasayco Maria Luisa, McDermott Ann, Cataldi Marcela, Arriaran Vilma, Polenova Tatyana
Department of Chemistry and Biochemistry, Brown Laboratories, University of Delaware, Newark, Delaware 19716, USA.
J Am Chem Soc. 2004 Dec 22;126(50):16608-20. doi: 10.1021/ja0464589.
De novo site-specific backbone and side-chain resonance assignments are presented for U-15N(1-73)/U-13C,15N(74-108) reassembly of Escherichia coli thioredoxin by fragment complementation, determined using solid-state magic angle spinning NMR spectroscopy at 17.6 T. Backbone dihedral angles and secondary structure predicted from the statistical analysis of 13C and 15N chemical shifts are in general agreement with solution values for the intact full-length thioredoxin, confirming that the secondary structure is retained in the reassembled complex prepared as a poly(ethylene glycol) precipitate. The differential labeling of complementary thioredoxin fragments introduced in this work is expected to be beneficial for high-resolution structural studies of protein interfaces formed by protein assemblies by solid-state NMR spectroscopy.
通过片段互补对大肠杆菌硫氧还蛋白进行U-15N(1-73)/U-13C,15N(74-108) 重新组装，利用17.6 T的固态魔角旋转核磁共振光谱确定了从头开始的位点特异性主链和侧链共振归属。通过对13C和15N化学位移的统计分析预测的主链二面角和二级结构，总体上与完整全长硫氧还蛋白的溶液值一致，证实二级结构保留在作为聚乙二醇沉淀制备的重新组装复合物中。这项工作中引入的互补硫氧还蛋白片段的差异标记，有望有利于通过固态核磁共振光谱对蛋白质组装形成的蛋白质界面进行高分辨率结构研究。
