Non-uniformly Sampled Double-TROSY hNcaNH Experiments for NMR Sequential Assignments of Large Proteins

详细信息    查看全文

• 作者：Dominique P. Frueh ; Zhen-Yu J. Sun ; David A. Vosburg ; Christopher T. Walsh ; Jeffrey C. Hoch ; and Gerhard Wagner
• 刊名：Journal of the American Chemical Society
• 出版年：2006
• 出版时间：May 3, 2006
• 年：2006
• 卷：128
• 期：17
• 页码：5757 - 5763
• 全文大小：210K
• 年卷期：v.128,no.17(May 3, 2006)
• ISSN：1520-5126

文摘

The initial step of protein NMR resonance assignments typically identifies the sequence positionsof ¹H-¹⁵N HSQC cross-peaks. This is usually achieved by tediously comparing strips of multiple triple-resonance experiments. More conveniently, this could be obtained directly with hNcaNH and hNcocaNH-type experiments. However, in large proteins and at very high fields, rapid transverse relaxation severelylimits the sensitivity of these experiments, and the limited spectral resolution obtainable in conventionallyrecorded experiments leaves many assignments ambiguous. We have developed alternative hNcaNHexperiments that overcome most of these limitations. The TROSY technique was implemented forsemiconstant time evolutions in both indirect dimensions, which results in remarkable sensitivity andresolution enhancements. Non-uniform sampling in both indirect dimensions combined with MaximumEntropy (MaxEnt) reconstruction enables such dramatic resolution enhancement while maintaining shortmeasuring times. Experiments are presented that provide either bidirectional or unidirectional connectivities.The experiments do not involve carbonyl coherences and thus do not suffer from fast chemical shiftanisotropy-mediated relaxation otherwise encountered at very high fields. The method was applied to a300 M sample of a 37 kDa fragment of the E. coli enterobactin synthetase module EntF, for which high-resolution spectra with an excellent signal-to-noise ratio were obtained within 4 days each.