Kumar Dinesh
Centre of Biomedical Magnetic Resonance, SGPGIMS Campus, Raibareli Road, Lucknow 226014, Uttar Pradesh, India.
J Struct Funct Genomics. 2013 Sep;14(3):109-18. doi: 10.1007/s10969-013-9161-y. Epub 2013 Aug 27.
Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone ((1)H, (15)N, (13)C(α) and (13)C') resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment--(4,3)D-hnCOCANH and exploits the linear combinations of backbone ((13)C(α) and (13)C') chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks--which is different in sum (CA + CO) and difference (CA - CO) frequency regions--greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide (15)N and backbone (13)C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (i→i + 1) (15)N and (13)C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F₂-F₃ planes of the spectrum corresponding to sum (CA + CO) and difference (CA - CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW < 15 kDa) or a range of intrinsically disordered proteins.
蛋白质的序列特异性共振归属是通过核磁共振进行各种结构和功能蛋白质组学研究的基础。在此背景下，本文提出了一种高效的独立方法，用于快速归属蛋白质的主链（（1）H、（15）N、（13）C（α）和（13）C'）共振。与目前用于此目的的现有策略相比，该方法仅采用一个降维实验——（4,3）D-hnCOCANH，并利用主链（（13）C（α）和（13）C'）化学位移的线性组合，以实现与单个化学位移相比相对更好的分散性（见正文）。所得峰的分散性增加——在总和（CA + CO）和差值（CA - CO）频率区域有所不同——通过解决由于简并酰胺（15）N和主链（13）C化学位移引起的问题（与常规归属策略相关），极大地促进了光谱分析。此外，由于峰的符号相反，该光谱可直接区分残基内和残基间的相关性。该光谱的另一个有益特征是它提供：（a）多个单向序列（i→i + 1）（15）N和（13）C相关性，以及（b）易于识别某些特定的三联体序列，这些序列可作为将连续连接的HSQC交叉峰的片段映射到一级序列以进行序列特异性共振归属的检查点。最重要的是，对应于总和（CA + CO）和差值（CA - CO）化学位移的光谱的F₂ - F₃平面通过匹配它们在这两个平面中的坐标，能够快速且明确地识别连续的HSQC峰（见正文）。总体而言，本文提出的实验将作为一种重要的主链归属工具，用于通过核磁共振进行的各种结构和功能蛋白质组学以及药物发现研究项目，这些项目涉及行为良好的小折叠蛋白（MW < 15 kDa）或一系列内在无序蛋白。
