Marulanda Dabeiba, Tasayco Maria Luisa, Cataldi Marcela, Arriaran Vilma, Polenova Tatyana
Department of Chemistry and Biochemistry, Brown Laboratories, University of Delaware, Newark, Delaware 19716, USA.
J Phys Chem B. 2005 Sep 29;109(38):18135-45. doi: 10.1021/jp052774d.
De novo site-specific 13C and 15N backbone and sidechain resonance assignments are presented for uniformly enriched E. coli thioredoxin, established using two-dimensional homo- and heteronuclear solid-state magic angle spinning NMR correlation spectroscopy. Backbone dihedral angles and secondary structure were derived from the statistical analysis of the secondary chemical shifts, and are in good agreement with solution values for the intact full-length thioredoxin, with the exception of a small number of residues located at the termini of the individual secondary structure elements. A large number of cross-peaks observed in the DARR spectra with long mixing times correspond to the pairs of carbon atoms separated by 4-6 angstroms, suggesting that DARR could be efficiently employed for observation of medium- and long-range correlations. The 108 amino acid residue E. coli thioredoxin is the largest uniformly enriched protein assigned to this degree of completeness by solid-state NMR spectroscopy to date. It is anticipated that with a combination of two-dimensional correlation experiments and high magnetic fields, resonance assignments and secondary structure can be generally derived for other noncrystalline proteins.
本文给出了均匀富集的大肠杆菌硫氧还蛋白从头进行的位点特异性13C和15N主链及侧链共振归属，这些归属是利用二维同核和异核固态魔角旋转NMR相关光谱法确定的。主链二面角和二级结构是从二级化学位移的统计分析中得出的，除了位于各个二级结构元件末端的少数残基外，它们与完整全长硫氧还蛋白的溶液值高度吻合。在具有长混合时间的DARR光谱中观察到的大量交叉峰对应于相隔4-6埃的碳原子对，这表明DARR可有效地用于观察中远程相关性。108个氨基酸残基的大肠杆菌硫氧还蛋白是迄今为止通过固态NMR光谱法在这种完整程度上归属的最大的均匀富集蛋白。预计结合二维相关实验和高磁场，其他非晶态蛋白质的共振归属和二级结构通常也可以推导出来。
