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.

中文翻译：

---

通过全局HDX-MS探索去污剂对完整膜蛋白LeuT构象的影响。

神经递质：钠共转运蛋白（NSS）是整合膜蛋白（IMP），负责从突触间隙再摄取神经递质。由于生产活性形式的哺乳动物NSS所面临的挑战，原核疏水氨基酸转运蛋白LeuT在这里用作阐明结构与功能关系的坚定模型。由于NSS蛋白位于磷脂双层中，因此提取时需要通过人工膜系统进行稳定。正确选择人工膜系统至关重要，因为次优的去污剂和/或脂质会导致不稳定或非天然稳定。在这里，我们通过全球HDX-MS研究了相关清洁剂十二烷基麦芽糖苷（DDM）和月桂基麦芽糖新戊二醇（LMNG）对LeuT构象动力学的影响，在功能上相关的配体存在下。我们观察到，与LMNG相比，LeuT在DDM中溶解时更具活力。此外，在K存在下LeuT显示出增加的HDX⁺相比的Na ⁺，表明K的存在的更可动态构象⁺。添加亮氨酸后，相对于Na ⁺结合状态，LeuT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/重折叠动力学。这些慢速动力学已通过本地HDX进行了验证，还证明了清洁剂可调节这些动力学的速率。

意义

总的来说，我们显示了整体HDX-MS评估人工膜系统对整体膜蛋白的影响的功效，以及为此类蛋白的增溶精心选择兼容的去污剂（和/或脂质）的重要性。