Probing the conformational impact of detergents on the integral membrane protein LeuT by global HDX-MS
| Autor: | Kasper D. Rand, Claus J. Loland, Ingvar R. Möller, Patrick S. Merkle, Solveig G. Schmidt, Dionisie Calugareanu, Gerard Comamala |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
030102 biochemistry & molecular biology Synaptic cleft Chemistry Detergents Molecular Conformation Biophysics Synthetic membrane Membrane Proteins Hydrogen Deuterium Exchange-Mass Spectrometry Biochemistry 03 medical and health sciences 030104 developmental biology Membrane protein Symporter Animals Hydrogen–deuterium exchange Amino acid transporter Maltose Lipid bilayer Integral membrane protein |
| Zdroj: | Journal of Proteomics. 225:103845 |
| ISSN: | 1874-3919 |
| DOI: | 10.1016/j.jprot.2020.103845 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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