Centre of Biomedical Research (CBMR), Sanjay Gandhi Post-Graduate Institute of Medical Sciences Campus, Raibareli Road, Lucknow 226014, Uttar Pradesh, India.
J Magn Reson. 2013 Dec;237:85-91. doi: 10.1016/j.jmr.2013.09.015. Epub 2013 Oct 8.
Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone (15)N and (13)C'/(13)C(α) chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone (15)N(i) chemical shifts with that of (13)C' (i-1)/(13)C(α) (i-1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate (15)N chemical shifts and (ii) reduces the signal overlap in F2((15)N)-F3((1)H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively.
提出了两种新的降维（RD） tailor HN(C)N [S.C. Panchal，N.S. Bhavesh，R.V. Hosur，改进的 3D 三重共振实验，HNN 和 HN(C)N，用于（13C，15N）标记蛋白中的 HN 和 15N 顺序相关：在 unfolded 蛋白中的应用，J. Biomol. NMR 20（2001）135-147]实验，以提高蛋白质的残基共振分配的准确性和速度。这些实验 - 在这里称为（4,3）D-hNCOcaNH 和（4,3）D-hNcoCANH - 利用同时组合 backbone（15）N 和（13）C' /（13）C（α）化学位移来实现更高的峰分散和沿其各自 F1 维度的随机性。简单地说，这是通过调制 backbone（15）N（i）化学位移与（13）C'（i-1）/（13）C（α）（i-1）自旋的化学位移来实现的，遵循已建立的降维 NMR 方法 [T. Szyperski，D.C. Yeh，D.K. Sukumaran，H.N. Moseley，G.T. Montelione，用于高通量蛋白共振分配的降维 NMR 光谱学，Proc. Natl. Acad. Sci. USA 99（2002）8009-8014]。虽然这种修饰很简单，但它在 HN(C)N 的峰分散和建立顺序连接的难易程度方面都取得了巧妙的改进。F1 维度上的分散度增加解决了两个目的：（i）解决了由于简并（15）N 化学位移引起的歧义，（ii）减少了 F2（（15）N）-F3（（1）H）平面中的信号重叠（在基于 HN（C）N 的分配协议中，这是一种重要的要求，用于方便且明确地识别顺序连接的 HSQC 峰）。这两种实验和分配方案的性能均使用牛脱钙 calbindin-d9k（75 个氨基酸）和脲变性 UNC60B（秀丽隐杆线虫 ADF/cofilin 家族的 152 个氨基酸蛋白）进行了证明，分别作为折叠蛋白和 unfolded 蛋白系统的代表。
