Vosegaard Thomas, Nielsen Niels C
Interdisciplinary Nanoscience Center (iNANO) and Laboratory for Biomolecular NMR Spectroscopy, Department of Molecular Biology, University of Aarhus, Aarhus, Denmark.
Magn Reson Chem. 2004 Feb;42(2):285-90. doi: 10.1002/mrc.1339.
Spin-exchange experiments are useful for improving the resolution and establishment of sequential assignments in solid-state NMR spectra of uniformly (15)N-labeled proteins oriented macroscopically in phospholipid bilayers. To exploit this advantage fully, it is crucial that the diagonal peaks in the two-dimensional exchange spectra are suppressed. This may be accomplished using the recent pure-exchange (PUREX) experiments, which, however, suffer from up to a threefold reduction of the cross-peak intensity relative to experiments without diagonal-peak suppression. This loss in sensitivity may severely hamper the applicability for the study of membrane proteins. In this paper, we present a two-dimensional exchange experiment (iPUREX) which improves the PUREX sensitivity by 50%. The performance of iPUREX is demonstrated experimentally by proton-mediated (15)N-(15)N spin-exchange experiments for a (15)N-labeled N-acetyl-L-valyl-L-leucine dipeptide. The relevance of exchange experiments with diagonal-peak suppression for large, uniformly (15)N-labeled membrane proteins in oriented phospholipid bilayers is demonstrated numerically for the G-protein coupled receptor rhodopsin.
自旋交换实验对于提高在磷脂双层中宏观取向的均匀（15）N 标记蛋白质的固态 NMR 谱的分辨率和进行序列归属至关重要。为了充分利用这一优势，二维交换谱中的对角峰被抑制至关重要。这可以通过最近的纯交换（PUREX）实验来实现，然而，相对于没有对角峰抑制的实验，其交叉峰强度最多降低了三倍。这种灵敏度的损失可能会严重阻碍其在膜蛋白研究中的适用性。在本文中，我们提出了一种二维交换实验（iPUREX），它将 PUREX 的灵敏度提高了 50%。通过质子介导的（15）N-（15）N 自旋交换实验对（15）N 标记的 N-乙酰-L-缬氨酰-L-亮氨酸二肽进行实验，证明了 iPUREX 的性能。对于 G 蛋白偶联受体视紫红质，通过数值模拟证明了在取向磷脂双层中对大的、均匀（15）N 标记的膜蛋白进行有对角峰抑制的交换实验的相关性。
