Jehle Stefan, Rehbein Kristina, Diehl Anne, van Rossum Barth-Jan
Leibniz-Institut für molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, D-13125 Berlin, Germany.
J Magn Reson. 2006 Dec;183(2):324-8. doi: 10.1016/j.jmr.2006.08.015. Epub 2006 Sep 20.
Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the alpha-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C-13C lysine-only correlation experiment.
氨基酸选择性魔角旋转（MAS）核磁共振实验有助于在固体蛋白质拥挤的光谱中确定模糊的交叉峰。特别是对于较大的蛋白质，数据分析可能会受到严重共振重叠的阻碍。在这种情况下，过滤技术可能是位点特异性自旋标记的一个很好的替代方法，以获得明确的归属，这些归属可以作为归属过程的起点。在本文中，我们提出了一个简单的脉冲序列，该序列允许对精氨酸和赖氨酸残基进行选择性激发。为了实现这一点，我们利用了选择性激发的特定交叉极化[M. 巴尔杜斯，A.T. 佩特科娃，J. 赫茨费尔德，R.G. 格里芬，倾斜框架中的交叉极化：异核自旋系统中的归属和光谱简化，《分子物理学》95（1998）1197 - 1207。]和沿着氨基酸侧链转移的自旋扩散的组合。通过在α - 血影蛋白SH3结构域的均匀13C和15N标记的蛋白质制备物中激发赖氨酸和精氨酸侧链共振，证明了该过滤器的选择性。结果表明，该过滤器可以作为仅赖氨酸的13C - 13C相关实验中的一个构建模块应用。
