TY - JOUR

T1 - Probing the conformational impact of detergents on the integral membrane protein LeuT by global HDX-MS

AU - Möller, Ingvar R

AU - Merkle, Patrick S

AU - Calugareanu, Dionisie

AU - Comamala, Gerard

AU - Schmidt, Solveig Gaarde

AU - Loland, Claus J

AU - Rand, Kasper D

N1 - Copyright © 2019. Published by Elsevier B.V.

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

U2 - 10.1016/j.jprot.2020.103845

DO - 10.1016/j.jprot.2020.103845

M3 - Journal article

C2 - 32480080

VL - 225

JO - Journal of Proteomics

JF - Journal of Proteomics

SN - 1874-3919

M1 - 103845

ER -