Battistel Marcos D, Azurmendi Hugo F, Yu Bingwu, Freedberg Darón I
Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20852-1448, United States.
Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20852-1448, United States.
Prog Nucl Magn Reson Spectrosc. 2014 May;79:48-68. doi: 10.1016/j.pnmrs.2014.01.001. Epub 2014 Feb 14.
The diversity in molecular arrangements and dynamics displayed by glycans renders traditional NMR strategies, employed for proteins and nucleic acids, insufficient. Because of the unique properties of glycans, structural studies often require the adoption of a different repertoire of tailor-made experiments and protocols. We present an account of recent developments in NMR techniques that will deepen our understanding of structure-function relations in glycans. We open with a survey and comparison of methods utilized to determine the structure of proteins, nucleic acids and carbohydrates. Next, we discuss the structural information obtained from traditional NMR techniques like chemical shifts, NOEs/ROEs, and coupling-constants, along with the limitations imposed by the unique intrinsic characteristics of glycan structure on these approaches: flexibility, range of conformers, signal overlap, and non-first-order scalar (strong) coupling. Novel experiments taking advantage of isotopic labeling are presented as an option for overcoming spectral overlap and raising sensitivity. Computational tools used to explore conformational averaging in conjunction with NMR parameters are described. In addition, recent developments in hydroxyl detection and hydrogen bond detection in protonated solvents, in contrast to traditional sample preparations in D2O for carbohydrates, further increase the tools available for both structure information and chemical shift assignments. We also include previously unpublished data in this context. Accurate determination of couplings in carbohydrates has been historically challenging due to the common presence of strong-couplings. We present new strategies proposed for dealing with their influence on NMR signals. We close with a discussion of residual dipolar couplings (RDCs) and the advantages of using (13)C isotope labeling that allows gathering one-bond (13)C-(13)C couplings with a recently improved constant-time COSY technique, in addition to the commonly measured (1)H-(13)C RDCs.
聚糖所展现出的分子排列和动力学的多样性使得用于蛋白质和核酸研究的传统核磁共振策略不再适用。由于聚糖具有独特的性质，结构研究往往需要采用一系列不同的定制实验和方案。我们介绍了核磁共振技术的最新进展，这些进展将加深我们对聚糖结构与功能关系的理解。我们首先对用于确定蛋白质、核酸和碳水化合物结构的方法进行了综述和比较。接下来，我们讨论了从传统核磁共振技术中获得的结构信息，如化学位移、核Overhauser效应/旋转Overhauser效应（NOEs/ROEs）以及耦合常数，同时也讨论了聚糖结构的独特内在特性给这些方法带来的局限性：灵活性、构象体范围、信号重叠以及非一级标量（强）耦合。利用同位素标记的新型实验被作为克服光谱重叠和提高灵敏度的一种选择进行了介绍。描述了结合核磁共振参数用于探索构象平均的计算工具。此外，与传统的用于碳水化合物的重水（D2O）样品制备相比，质子化溶剂中羟基检测和氢键检测的最新进展进一步增加了可用于获取结构信息和化学位移归属的工具。在此背景下，我们还纳入了之前未发表的数据。由于强耦合普遍存在，碳水化合物中耦合的准确测定在历史上一直具有挑战性。我们介绍了为应对其对核磁共振信号的影响而提出的新策略。最后，我们讨论了残余偶极耦合（RDCs）以及使用（13）C同位素标记的优势，除了常用的测量（1）H - （13）C RDCs外，这种标记还能通过最近改进的恒时COSY技术收集一键（13）C - （13）C耦合。
