Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen O, Denmark.
Laboratory for Membrane Protein Dynamics, Department of Neuroscience, University of Copenhagen, 2200 Copenhagen N, Denmark.
J Proteomics. 2020 Aug 15;225:103845. doi: 10.1016/j.jprot.2020.103845. Epub 2020 May 30.
Neurotransmitter:sodium symporters (NSS) are integral membrane proteins (IMP), responsible for reuptake of neurotransmitters from the synaptic cleft. Due to challenges in production of mammalian NSS in their active form, the prokaryotic hydrophobic amino acid transporter, LeuT, served here as a steadfast model for elucidation of structure-function relationship. As NSS proteins reside within phospholipid bilayer, they require stabilization by artificial membrane systems upon their extraction. Right choice of artificial membrane system is crucial as suboptimal detergent and/or lipids can lead to destabilization or non-native stabilization. Here we study the effect of related detergents, dodecyl maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG), on the conformational dynamics of LeuT by global HDX-MS, in the presence of functionally relevant ligands. We observed that LeuT is more dynamic when solubilized in DDM compared to LMNG. Moreover, LeuT exhibited increased HDX in the presence of K compared to Na, indicating a more dynamic conformation in the presence of K. Upon addition of leucine, LeuT underwent additional stabilization relative to the Na-bound state. Finally, peak broadening was observed, suggesting that LeuT undergoes slow unfolding/refolding dynamics in detergent solution. These slow dynamics were verified by local HDX, also proving that detergents modulate the rate of these dynamics. SIGNIFICANCE: Overall, we show the efficacy of global HDX-MS to evaluate the effect of artificial membrane systems on integral membrane proteins and the importance of carefully selecting compatible detergent (and/or lipid) for the solubilization of this class of proteins.
钠协同转运蛋白（NSS）是一种整合膜蛋白（IMP），负责从突触间隙中重新摄取神经递质。由于在哺乳动物 NSS 的活性形式的生产中存在挑战，因此原核疏水性氨基酸转运蛋白 LeuT 在这里作为阐明结构-功能关系的坚定模型。由于 NSS 蛋白存在于磷脂双层中，因此在提取后需要人工膜系统来稳定它们。人工膜系统的正确选择至关重要，因为不合适的去污剂和/或脂质可能导致不稳定或非天然稳定。在这里，我们通过全局 HDX-MS 研究了相关去污剂十二烷基麦芽糖（DDM）和月桂基麦芽糖新戊二醇（LMNG）对功能相关配体存在下 LeuT 构象动力学的影响。我们观察到，与 LMNG 相比，LeuT 在 DDM 中更具动态性。此外，与 Na 相比，LeuT 在存在 K 时表现出更高的 HDX，表明在 K 存在下存在更具动态性的构象。当加入亮氨酸时，LeuT 相对于 Na 结合态经历了额外的稳定化。最后，观察到峰展宽，表明 LeuT 在去污剂溶液中经历缓慢的展开/折叠动力学。这些缓慢的动力学通过局部 HDX 得到了验证，也证明了去污剂调节了这些动力学的速率。意义：总的来说，我们展示了全局 HDX-MS 评估人工膜系统对整合膜蛋白的影响的效果，以及仔细选择兼容去污剂（和/或脂质）对这类蛋白质的溶解的重要性。
