Murray Dylan T, Li Conggang, Gao F Philip, Qin Huajun, Cross Timothy A
National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida; Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida.
State Key Laboratory of Magnetic Resonance and Molecular and Atomic Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, PR China.
Biophys J. 2014 Apr 15;106(8):1559-69. doi: 10.1016/j.bpj.2014.02.026.
The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.
多年来，通过功能测定来验证蛋白质结构一直是常规做法。功能测定对于水溶性蛋白质的这种验证效果很好，但它们需要在与结构分析相同的环境中进行。对于通常在去污剂环境中进行结构表征的膜蛋白来说，这很难做到，尽管这些蛋白质的功能测定最常在脂质双层中进行。由于已知膜蛋白的结构对膜模拟环境敏感，这种功能测定适用于验证蛋白质构建体，但不适用于验证膜蛋白结构。在这里，我们将先前发表的二酰基甘油激酶的定向样品固态核磁共振光谱数据与基于该蛋白质近期结构对这类数据的预测进行了比较。已在去污剂胶束中获得了二酰基甘油激酶的溶液核磁共振结构，并在单油酸立方相中获得了三种晶体结构。所有结构均为三聚体，每个单体具有三个跨膜螺旋和一个两亲性螺旋。然而，溶液核磁共振结构显示出由胶束环境引起的典型扰动，这反映在从结构坐标预测的固态核磁共振共振中。晶体结构显示出很少有这样的扰动，特别是对于野生型结构，尤其是对于没有显著晶体接触的单体。对于这些单体，预测数据和观测数据几乎相同。热稳定构建体确实显示出更多的扰动，特别是A41C突变，该突变将一个亲水残基引入到脂质双层中间位置，导致三聚体之间形成额外的氢键。这些结果证明了一种通用技术，可通过定向样品固态核磁共振，利用从液晶脂质双层中的膜蛋白获得的最少数据来验证膜蛋白结构。
