Milbradt Alexander G, Arthanari Haribabu, Takeuchi Koh, Boeszoermenyi Andras, Hagn Franz, Wagner Gerhard
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA.
J Biomol NMR. 2015 Jul;62(3):291-301. doi: 10.1007/s10858-015-9944-5. Epub 2015 May 10.
For typical globular proteins, contacts involving aromatic side chains would constitute the largest number of distance constraints that could be used to define the structure of proteins and protein complexes based on NOE contacts. However, the (1)H NMR signals of aromatic side chains are often heavily overlapped, which hampers extensive use of aromatic NOE cross peaks. Some of this overlap can be overcome by recording (13)C-dispersed NOESY spectra. However, the resolution in the carbon dimension is rather low due to the narrow dispersion of the carbon signals, large one-bond carbon-carbon (C-C) couplings, and line broadening due to chemical shift anisotropy (CSA). Although it has been noted that the CSA of aromatic carbons could be used in TROSY experiments for enhancing resolution, this has not been used much in practice because of complications arising from large aromatic one-bond C-C couplings, and 3D or 4D carbon dispersed NOESY are typically recorded at low resolution hampering straightforward peak assignments. Here we show that the aromatic TROSY effect can optimally be used when employing alternate (13)C labeling using 2-(13)C glycerol, 2-(13)C pyruvate, or 3-(13)C pyruvate as the carbon source. With the elimination of the strong one-bond C-C coupling, the TROSY effect can easily be exploited. We show that (1)H-(13)C TROSY spectra of alternately (13)C labeled samples can be recorded at high resolution, and we employ 3D NOESY aromatic-TROSY spectra to obtain valuable intramolecular and intermolecular cross peaks on a protein complex.
对于典型的球状蛋白质，基于核Overhauser效应（NOE）接触来定义蛋白质和蛋白质复合物结构时，涉及芳香族侧链的接触将构成可使用的距离约束的最大数量。然而，芳香族侧链的（1）H NMR信号常常严重重叠，这妨碍了芳香族NOE交叉峰的广泛使用。通过记录（13）C 分散的NOESY光谱可以克服部分这种重叠。然而，由于碳信号的色散较窄、一键碳 - 碳（C - C）耦合较大以及化学位移各向异性（CSA）导致的线展宽，碳维度的分辨率相当低。尽管已经注意到芳香族碳的CSA可用于横向弛豫优化谱（TROSY）实验以提高分辨率，但由于大的芳香族一键C - C耦合产生的复杂性，在实践中这并未得到太多应用，并且3D或4D碳分散NOESY通常在低分辨率下记录，妨碍了直接的峰归属。在这里我们表明，当使用2 - （13）C甘油、2 - （13）C丙酮酸或3 - （13）C丙酮酸作为碳源进行交替（13）C标记时，可以最佳地利用芳香族TROSY效应。随着强一键C - C耦合的消除，可以轻松利用TROSY效应。我们表明，可以在高分辨率下记录交替（13）C标记样品的（1）H - （13）C TROSY光谱，并且我们使用3D NOESY芳香族 - TROSY光谱在蛋白质复合物上获得有价值的分子内和分子间交叉峰。
