Cross T A
Center for Interdisciplinary Magnetic Resonance, Florida State University, Talahassee 32306-4005, USA.
Methods Enzymol. 1997;289:672-96. doi: 10.1016/s0076-6879(97)89070-2.
The method of using orientational constraints derived from solid-state NMR for structural characterization of polypeptides in heterogeneous environments has now been demonstrated. A very high resolution structure has been achieved that has led to greater functional understanding of this channel. Much can be done to improve this structural technique to make it more efficient and more generally applicable. Others as well as ourselves are applying this approach to membrane proteins. Although solid-phase synthesis and specific site isotopic labeling has been essential for the development described here, one of the primary challenges is to be able to use amino acid-specific and uniform labeling of peptides and proteins by biosynthetic means for isotopic incorporation. This will allow for the study of many more proteins and significantly large proteins. Unlike solution NMR structural methods, there are no intrinsic molecular weight limitations. In fact, as the molecular weight increases the molecular motion will become less and the spectroscopic properties will improve. The major limitation will be sensitivity: as the molecular weight increases the number of moles will decrease in the samples, causing sensitivity to decrease. Advances in field strength and NMR technology help to address this problem. With larger molecules and more isotopically labeled sites resolution could also be a problem; however, the two- and three-dimensional methods demonstrated by Opella and co-workers clearly show the potential for enormous resolving power. In the 15N dimension alone it is shown that the resolution is greater than in solution NMR. Although challenges such as spectral assignments have yet to be completely solved, several approaches have been described, and the prospects are excellent for solving this and other problems facing the development of this novel approach for structural elucidation. Although there is an attempt to get away from solid-phase synthesis to solve larger molecular weight structures, peptide synthesis will continue to be important for generating single- and double-site labeled model compounds for characterizations of spin interaction tensors. Such characterizations will continue to be a very important aspect of this structural approach.
利用固态核磁共振得出的取向限制对异质环境中的多肽进行结构表征的方法现已得到证实。已获得了非常高分辨率的结构，这使得对该通道的功能有了更深入的了解。在改进这种结构技术以使其更高效、更广泛适用方面还有很多工作要做。包括我们自己在内的其他人正在将这种方法应用于膜蛋白。尽管固相合成和特定位点同位素标记对于此处所述的进展至关重要，但主要挑战之一是能够通过生物合成手段对肽和蛋白质进行氨基酸特异性和均匀标记以实现同位素掺入。这将有助于研究更多的蛋白质以及显著更大的蛋白质。与溶液核磁共振结构方法不同，不存在内在的分子量限制。事实上，随着分子量增加，分子运动将减少，光谱性质将得到改善。主要限制将是灵敏度：随着分子量增加，样品中的摩尔数将减少，导致灵敏度降低。场强和核磁共振技术的进步有助于解决这个问题。对于更大的分子和更多的同位素标记位点，分辨率也可能是个问题；然而，奥佩拉及其同事展示的二维和三维方法清楚地显示了巨大的分辨能力的潜力。仅在15N维度就表明分辨率高于溶液核磁共振。尽管诸如光谱归属等挑战尚未完全解决，但已经描述了几种方法，并且解决这一新型结构解析方法发展中面临的这一问题和其他问题的前景非常好。尽管有人试图摆脱固相合成以解决更大分子量的结构，但肽合成对于生成用于自旋相互作用张量表征的单位点和双位点标记模型化合物仍将继续发挥重要作用。这种表征将继续是这种结构方法的一个非常重要的方面。
