Frueh Dominique P, Sun Zhen-Yu J, Vosburg David A, Walsh Christopher T, Hoch Jeffrey C, Wagner Gerhard
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
J Am Chem Soc. 2006 May 3;128(17):5757-63. doi: 10.1021/ja0584222.
The initial step of protein NMR resonance assignments typically identifies the sequence positions of 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 severely limits the sensitivity of these experiments, and the limited spectral resolution obtainable in conventionally recorded experiments leaves many assignments ambiguous. We have developed alternative hNcaNH experiments that overcome most of these limitations. The TROSY technique was implemented for semiconstant time evolutions in both indirect dimensions, which results in remarkable sensitivity and resolution enhancements. Non-uniform sampling in both indirect dimensions combined with Maximum Entropy (MaxEnt) reconstruction enables such dramatic resolution enhancement while maintaining short measuring 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 shift anisotropy-mediated relaxation otherwise encountered at very high fields. The method was applied to a 300 microM 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.
蛋白质核磁共振共振归属的初始步骤通常是确定1H-15N HSQC交叉峰的序列位置。这通常是通过繁琐地比较多个三重共振实验的条带实现的。更方便的是，这可以直接通过hNcaNH和hNcocaNH型实验获得。然而，在大蛋白质和非常高的场强下，快速横向弛豫严重限制了这些实验的灵敏度，并且传统记录实验中可获得的有限光谱分辨率使得许多归属不明确。我们开发了替代的hNcaNH实验，克服了这些限制中的大部分。TROSY技术用于两个间接维度的半恒定时间演化，这导致了显著的灵敏度和分辨率增强。两个间接维度的非均匀采样与最大熵（MaxEnt）重建相结合，在保持测量时间短的同时实现了如此显著的分辨率增强。展示了提供双向或单向连接性的实验。这些实验不涉及羰基相干，因此不会受到在非常高的场强下否则会遇到的快速化学位移各向异性介导的弛豫的影响。该方法应用于大肠杆菌肠杆菌素合成酶模块EntF的37 kDa片段的300 microM样品，在4天内分别获得了具有出色信噪比的高分辨率光谱。
