Department for NMR-based Structural Biology , Max Planck Institute for Biophysical Chemistry , Am Faßberg 11 , 37077 Göttingen , Germany.
Institute of Cellular and Molecular Immunology, Georg August University of Göttingen , Humboldtallee 34 , 37073 Göttingen , Germany.
J Am Chem Soc. 2018 Mar 14;140(10):3518-3522. doi: 10.1021/jacs.8b00215. Epub 2018 Mar 6.
Proline is prevalent in intrinsically disordered proteins (IDPs). NMR assignment of proline-rich IDPs is a challenge due to low dispersion of chemical shifts. We propose here new sensitivity-enhanced 4D NMR experiments that correlate two pairs of amide resonances that are either consecutive (NH , NH ) or flanking a proline at position i-1 (NH , NH ). The maximum 2-fold enhancement of sensitivity is achieved by employing two coherence order-selective (COS) transfers incorporated unconventionally into the pulse sequence. Each COS transfer confers an enhancement over amplitude-modulated transfer by a factor of √2 specifically when transverse relaxation is slow. The experiments connect amide resonances over a long fragment of sequence interspersed with proline. When this method was applied to the proline-rich region of B cell adaptor protein SLP-65 (pH 6.0) and α-synuclein (pH 7.4), which contain a total of 52 and 5 prolines, respectively, 99% and 92% of their nonprolyl amide resonances have been successfully assigned, demonstrating its robustness to address the assignment problem in large proline-rich IDPs.
脯氨酸在无规则卷曲蛋白质（IDP）中很常见。由于化学位移的分散度低，富含脯氨酸的 IDP 的 NMR 分配是一个挑战。我们在这里提出了新的灵敏度增强的 4D NMR 实验，这些实验可以关联两个酰胺共振对，要么是连续的（NH, NH ），要么是位于位置 i-1 的脯氨酸的侧翼（NH, NH ）。通过在脉冲序列中非常规地采用两个相干性顺序选择（COS）转移，可以实现最大 2 倍的灵敏度增强。每个 COS 转移通过因子√2 赋予比幅度调制转移更高的增强，特别是当横向弛豫缓慢时。这些实验将富含脯氨酸的区域与富含脯氨酸的区域连接起来序列片段，其中包含总共 52 个和 5 个脯氨酸。当将此方法应用于 B 细胞衔接蛋白 SLP-65（pH 6.0）和α-突触核蛋白（pH 7.4）的富含脯氨酸区域时，成功地分配了它们的非脯氨酸酰胺共振的 99%和 92%，证明了其在解决大型富含脯氨酸 IDP 的分配问题方面的稳健性。
