固体核磁共振中膜蛋白双交叉极化效率与动力学参数相关的定量分析

摘要

固体核磁共振(NMR)中双交叉极化(DCP)是用于膜蛋白信号指认的多维异核相关实验的基本技术模块.DCP的效率在很大程度上决定了多维异核相关实验的效率.本文分析了3种典型的膜环境中的膜蛋白(AQPZ、DAGK和EV712B)的DCP效率及其影响因素.结果显示,在相同的实验条件下,3种蛋白样品的DCP效率存在明显差异:其中AQPZ的DCP效率最高(31%),DAGK的效率次之(23%),EV712B的效率最低(14%).通过测量它们在旋转坐标下的自旋-晶格弛豫时间(T1ρ)和偶极耦合常数(DHN),发现膜蛋白的运动会明显缩短T1ρ,但对DHN的影响较小.在实验的基础上,建立了T1ρ 与DCP效率相关的模型,并基于DCP动力学的定量分析,证明了运动导致的T1ρ 缩短是降低DCP效率的主要原因.因此,可以通过定量分析未知样品的T1ρ 来预测其DCP的最优效率,为DCP实验的优化提供依据.%The efficiency of multiple-dimensional heteronuclear correlation experiments in membrane protein studies is partially determined by the efficiency of double cross polarization (DCP). DCP is a basic block in the pulse sequences to probe the connections between the 15N and 13C nuclei. In this study, three membrane proteins were studied experimentally to determine how the dynamics properties of the proteins affect the DCP efficiency in solid-state heteronuclear correlation experiments. Under the same experimental conditions, the AQPZ protein showed the highest DCP efficiency (31%), while the EV712B protein had the lowest efficiency (14%) and the DAGK protein in between (23%). Longitudinal lattice relaxation time in the rotating frame (T1ρ) and dipole coupling constant (DHN) were also measured for these proteins. It was found that the DCP efficiency depended strongly on T1ρ, but only weakly on DHN. Based on the experimental data, a model to correlate the T1ρ of protein to the DCP efficiency was established, with which the DCP efficiency could be predicted by measuring the T1ρ of membrane proteins.