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dc.contributor.authorAdhikary, S.
dc.contributor.authorDeredge, D.J.
dc.contributor.authorNagarajan, A.
dc.date.accessioned2019-11-01T12:49:40Z
dc.date.available2019-11-01T12:49:40Z
dc.date.issued2017
dc.identifier.urihttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85014862382&doi=10.1073%2fpnas.1613293114&partnerID=40&md5=597f3dbe0c24a5b20edb9cbf398c202f
dc.identifier.urihttp://hdl.handle.net/10713/11353
dc.description.abstractNeurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of smallmolecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.en_US
dc.description.sponsorshipThis work was supported by the Alfred P. Sloan Foundation (S.K.S.); the Brain and Behavior Research Foundation (S.K.S.); a Goodman- Gilman Yale Scholar Award (to S.K.S.); NIH/National Institute of Mental Health Grants R00MH083050 (to S.K.S.) and R01MH100688 (to S.K.S.); the Division of Intramural Research of the NIH, National Institute of Neurological Disorders and Stroke (L.R.F.); and the University of Maryland Baltimore, School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014).en_US
dc.description.urihttps://doi.org/10.1073/pnas.1613293114en_US
dc.language.isoen_USen_US
dc.publisherNational Academy of Sciencesen_US
dc.relation.ispartofProceedings of the National Academy of Sciences of the United States of America
dc.subjectConformational dynamicsen_US
dc.subjectHydrogen-deuterium exchange mass spectrometryen_US
dc.subjectMolecular dynamics simulationsen_US
dc.subjectNanodiscen_US
dc.subjectNeurotransmitter symporteren_US
dc.titleConformational dynamics of a neurotransmitter: Sodium symporter in a lipid bilayeren_US
dc.typeArticleen_US
dc.identifier.doi10.1073/pnas.1613293114
dc.identifier.pmid28223522


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