Zhuang Xiaohong, Klauda Jeffery B
Department of Chemical and Biomolecular Engineering, College Park, MD 20742, USA.
Department of Chemical and Biomolecular Engineering, College Park, MD 20742, USA; Biophysics Program, University of Maryland, College Park, MD 20742, USA.
Biochim Biophys Acta. 2016 Jul;1858(7 Pt A):1541-52. doi: 10.1016/j.bbamem.2016.04.008. Epub 2016 Apr 20.
Lactose permease of E. coli (LacY) is a secondary active transporter (SAT) that belongs to the major facilitator superfamily (MFS). Experimental structures of the cytoplasmic-open and more recently occluded-like structure have been determined, however, the crystal structure of LacY in the periplasmic-open state is still not available. The periplasmic-open LacY structure is important for understanding complete proton/sugar transport process of LacY as well as other similar SAT proteins. Previously, a structural model of periplasmic-open LacY has been obtained through a two-step hybrid implicit-explicit (IM-EX) simulation method (JMB404: 506). Molecular dynamics simulations are performed to further test the IM-EX model for the periplasmic-open LacY with ββ-(Galp)2 in a lipid membrane. The comparison of the calculated pore radii to the data of the crystal structure indicates that the IM-EX model of LacY remains periplasmic-open in E269-protonated states. The neighbor residue distance change based on Cα are very similar in simulation results, but they are significantly different in double electron-electron resonance (DEER) experimental data, which motivates us to perform the molecular dynamics dummy spin-label (MDDS) simulations to test the effect of spin labels (size and internal flexibility) on DEER spin label distance measurements. The MDDS simulation results show that the orientation and movement of the spin labels significantly affect the residue pair distance measurement. DEER data alone may not provide an accurate guide for predicting protein structures. MDDS simulations can be applied to analyze the distance distribution due to spin labels and also aid in proper interpretation of DEER experimental data.
大肠杆菌乳糖通透酶（LacY）是一种属于主要易化子超家族（MFS）的次级主动转运蛋白（SAT）。目前已确定了其胞质开放状态以及最近类似堵塞状态的实验结构，然而，周质开放状态下LacY的晶体结构仍未获得。周质开放状态的LacY结构对于理解LacY以及其他类似SAT蛋白完整的质子/糖转运过程至关重要。此前，通过两步混合隐式-显式（IM-EX）模拟方法（《分子生物学杂志》404卷：506页）获得了周质开放状态LacY的结构模型。进行分子动力学模拟以进一步测试脂质膜中含ββ-(半乳糖)2的周质开放状态LacY的IM-EX模型。将计算得到的孔径半径与晶体结构数据进行比较表明，LacY的IM-EX模型在E269质子化状态下仍保持周质开放。基于Cα的相邻残基距离变化在模拟结果中非常相似，但在双电子-电子共振（DEER）实验数据中却有显著差异，这促使我们进行分子动力学虚拟自旋标记（MDDS）模拟，以测试自旋标记（大小和内部灵活性）对DEER自旋标记距离测量的影响。MDDS模拟结果表明，自旋标记的取向和运动会显著影响残基对距离测量。仅靠DEER数据可能无法为预测蛋白质结构提供准确指导。MDDS模拟可用于分析自旋标记引起的距离分布，也有助于对DEER实验数据进行正确解读。
