Bhavesh N S, Panchal S C, Hosur R V
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India.
Biochemistry. 2001 Dec 11;40(49):14727-35. doi: 10.1021/bi015683p.
Sequence specific resonance assignment is the primary requirement for all investigations of proteins by NMR methods. In the present postgenomic era where structural genomics and protein folding have occupied the center stage of NMR research, there is a high demand on the speed of resonance assignment, whereas the presently available methods based either on NOESY or on some triple-resonance experiments are rather slow. They also have limited success with unfolded proteins because of the lack of NOEs, and poor dispersion of amide and carbon chemical shifts. This paper describes an efficient approach to rapid resonance assignment that is suitable for both folded and unfolded proteins, making use of the triple-resonance experiments described recently [HNN and HN(C)N]. It has three underlying principles. First, the experiments exploit the (15)N chemical shift dispersions which are generally very good for both folded and unfolded proteins, along two of the three dimensions; second, they directly display sequential amide and (15)N correlations along the polypeptide chain, and third, the sign patterns of the diagonal and the sequential peaks originating from any residue are dependent on the nature of the adjacent residues, especially the glycines and the prolines. These lead to so-called "triplet fixed points" which serve as starting points and/or check points during the course of sequential walks, and explicit side chains assignment becomes less crucial for unambiguous backbone assignment. These features significantly enhance the speed of data analysis, reduce the amount of experimentation required, and thus result in a substantially faster and unambiguous assignment. Following the amide and (15)N assignments, the other proton and carbon assignments can be obtained in a straightforward manner, from the well-established three-dimensional triple-resonance experiments. We have successfully tested the new approach with different proteins in the molecular mass range of 10-22 kDa, and for illustration, we present here the backbone results on the HIV-1 protease-tethered dimer (molecular mass approximately 22 kDa), both in the folded and in the unfolded forms, the two ends of the folding funnel. We believe that the new assignment approach will be of great value for both structural genomics and protein folding research by NMR.
序列特异性共振归属是通过核磁共振方法对蛋白质进行所有研究的首要要求。在当前的后基因组时代，结构基因组学和蛋白质折叠已占据核磁共振研究的核心地位，对共振归属的速度有很高的要求，而目前基于NOESY或某些三共振实验的方法相当缓慢。由于缺乏NOE以及酰胺和碳化学位移的分散性较差，它们对未折叠蛋白质的成功程度也有限。本文描述了一种适用于折叠和未折叠蛋白质的快速共振归属的有效方法，该方法利用了最近描述的三共振实验[HNN和HN(C)N]。它有三个基本原理。首先，这些实验利用了通常对折叠和未折叠蛋白质都非常好的(15)N化学位移分散性，沿着三个维度中的两个维度；其次，它们直接显示沿着多肽链的连续酰胺和(15)N相关性，第三，源自任何残基的对角线和连续峰的符号模式取决于相邻残基的性质，特别是甘氨酸和脯氨酸。这些导致了所谓的“三联体固定点”，它们在连续步移过程中作为起点和/或检查点，并且对于明确的主链归属而言，明确的侧链归属变得不那么关键。这些特征显著提高了数据分析的速度，减少了所需的实验量，从而导致更快且明确的归属。在酰胺和(15)N归属之后，可以从成熟的三维三共振实验中以直接的方式获得其他质子和碳的归属。我们已经成功地用分子量在10 - 22 kDa范围内的不同蛋白质测试了这种新方法，并且为了说明，我们在此展示了HIV - 1蛋白酶连接二聚体（分子量约22 kDa）在折叠和未折叠形式下的主链结果，这是折叠漏斗的两端。我们相信这种新的归属方法对于核磁共振的结构基因组学和蛋白质折叠研究都将具有很大的价值。
