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

• Published in: Journal of the American Chemical Society Vol. 128; no. 17; pp. 5757 - 5763
• Main Authors: Frueh, Dominique P, Sun, Zhen-Yu J, Vosburg, David A, Walsh, Christopher T, Hoch, Jeffrey C, Wagner, Gerhard
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 03.05.2006
• Subjects: Biological and medical sciences; Chromatography, Gel; Fundamental and applied biological sciences. Psychology; Molecular biophysics; Nuclear Magnetic Resonance, Biomolecular - methods; Plasmids; Proteins - chemistry; Spectroscopy : techniques and spectras; Structure in molecular biology; Thermodynamics; Tridimensional structure; Nitrogen 15; Three dimensional structure; Molecular structure; Biological macromolecule; NMR spectrometry; Molecular connectivity; Heteronuclear correlation; Two dimension spectrometry; Pulse sequence
• ISSN: 0002-7863; 1520-5126
• 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 μM 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.