2023
Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer
Panda A, Giska F, Duncan A, Welch A, Brown C, McAllister R, Hariharan P, Goder J, Coleman J, Ramakrishnan S, Pincet F, Guan L, Krishnakumar S, Rothman J, Gupta K. Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer. Nature Methods 2023, 20: 891-897. PMID: 37106230, PMCID: PMC10932606, DOI: 10.1038/s41592-023-01864-5.Peer-Reviewed Original ResearchConceptsIntegral membrane proteinsMembrane proteinsOligomeric organizationOligomeric stateNative mass spectrometry analysisFunctional oligomeric stateKey membrane componentMass spectrometry analysisNMS analysisTarget membraneLipid bindingMembrane componentsProteolipid vesiclesMembrane compositionLipid compositionSpectrometry analysisLipid membranesNeurotransmitter releaseProteinMembraneLipidsMembrane propertiesDirect determinationBilayersTransporters
2022
Native Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip (Small 51/2022)
Sundaram R, Bera M, Coleman J, Weerakkody J, Krishnakumar S, Ramakrishnan S. Native Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip (Small 51/2022). Small 2022, 18 DOI: 10.1002/smll.202270277.Peer-Reviewed Original ResearchGiant plasma membrane vesiclesTotal internal reflection fluorescence microscopyMembrane protein assemblyPlasma membrane vesiclesReflection fluorescence microscopyDifferent cell typesSingle-molecule investigationsProtein functionProtein assembliesInner leafletPlasma membraneMembrane vesiclesCell typesLipid architectureFluorescence microscopyLipid membranesMolecule investigationsMembraneSilicon-based platformVesiclesAssemblyCellsBilayersLeafletsNative Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip
Sundaram R, Bera M, Coleman J, Weerakkody J, Krishnakumar S, Ramakrishnan S. Native Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip. Small 2022, 18: e2205567. PMID: 36328714, DOI: 10.1002/smll.202205567.Peer-Reviewed Original ResearchConceptsPlasma membraneProtein assembliesNative plasma membrane vesiclesTotal internal reflection fluorescence microscopySingle-molecule levelSingle-molecule investigationsCellular plasma membranePlasma membrane vesiclesSingle-molecule precisionReflection fluorescence microscopyKnowledge of lipidProtein complexesProtein architectureCell signalingMovement of moleculesProtein orientationLipid membranesBiological processesCellular membranesMembrane vesiclesMicroarray platformFluorescence investigationsLipid domainsFluorescence microscopyMembraneMolecular determinants of complexin clamping and activation function
Bera M, Ramakrishnan S, Coleman J, Krishnakumar SS, Rothman JE. Molecular determinants of complexin clamping and activation function. ELife 2022, 11: e71938. PMID: 35442188, PMCID: PMC9020821, DOI: 10.7554/elife.71938.Peer-Reviewed Original ResearchConceptsSynaptotagmin-1Single-vesicle fusionAccessory helixFusion clampHelical domainMolecular detailsComplexinMutational analysisVesicle releaseFusion kineticsMolecular determinantsSpecific interactionsInhibitory functionProbability of fusionRapid CaSNAREpinsAssembly processFusionClamping functionDomainHelixVesiclesFunctionMembraneInteractionA tunable lipid bilayer native MS platform for direct determination of hierarchical organization of membrane proteins and lipids at the membrane
Panda A, Giska F, Brown C, Coleman J, Rothman J, Gupta K. A tunable lipid bilayer native MS platform for direct determination of hierarchical organization of membrane proteins and lipids at the membrane. Biophysical Journal 2022, 121: 312a-313a. DOI: 10.1016/j.bpj.2021.11.1192.Peer-Reviewed Original Research
2019
Structural basis for the clamping and Ca2+ activation of SNARE-mediated fusion by synaptotagmin
Grushin K, Wang J, Coleman J, Rothman JE, Sindelar CV, Krishnakumar SS. Structural basis for the clamping and Ca2+ activation of SNARE-mediated fusion by synaptotagmin. Nature Communications 2019, 10: 2413. PMID: 31160571, PMCID: PMC6546687, DOI: 10.1038/s41467-019-10391-x.Peer-Reviewed Original ResearchConceptsCryo-electron microscopy structureActivation of SNAREsDependent membrane interactionsAnionic lipid headgroupsFusion clampActivator functionSNARE bundleSNARE proteinsMicroscopy structureC2B domainStructural basisSynaptotagmin-1SNAREpinsAliphatic loopsMembrane interactionsComplete assemblyLipid headgroupsLipid membranesNeurotransmitter releaseMembraneKey determinantSynaptotagminSyt1Calcium influxPartial insertionHighly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup
Heo P, Ramakrishnan S, Coleman J, Rothman JE, Fleury J, Pincet F. Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup. Small 2019, 15: e1900725. PMID: 30977975, DOI: 10.1002/smll.201900725.Peer-Reviewed Original ResearchConceptsMost biological processesLipid leafletAreas of biologyEmbedded proteinsBiological processesRelevant lipidsProteinAsymmetric bilayersPhysiological conditionsModel membranesPlanar bilayersBilayer formation processInvaluable insightsBilayersConfocal microscopeMembraneLipidsTransmembraneBiologyLeafletsMicrofluidic setupRecapitulation
2018
High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
Ramakrishnan S, Gohlke A, Li F, Coleman J, Xu W, Rothman JE, Pincet F. High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis. Langmuir 2018, 34: 5849-5859. PMID: 29694054, DOI: 10.1021/acs.langmuir.8b00116.Peer-Reviewed Original ResearchConceptsMembrane fusionFusion eventsSoluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteinsSNARE-dependent membrane fusionIndividual vesicle fusion eventsFactor attachment protein receptor proteinsN-ethylmaleimide-sensitive factor attachment protein receptor proteinsT-SNARE proteinsSingle-vesicle fusionProtein receptor proteinsVesicle fusion eventsMobility of proteinsVesicle dockingContent releaseVesicle fusionHigh-throughput monitoringPlanar membranesReceptor proteinLipid mixingProteinLipid bilayersVesiclesCorrect reconstitutionMembraneAqueous compartmentRearrangements under confinement lead to increased binding energy of Synaptotagmin‐1 with anionic membranes in Mg2+ and Ca2+
Gruget C, Coleman J, Bello O, Krishnakumar SS, Perez E, Rothman JE, Pincet F, Donaldson SH. Rearrangements under confinement lead to increased binding energy of Synaptotagmin‐1 with anionic membranes in Mg2+ and Ca2+. FEBS Letters 2018, 592: 1497-1506. PMID: 29578584, DOI: 10.1002/1873-3468.13040.Peer-Reviewed Original Research
2010
A fast, single-vesicle fusion assay mimics physiological SNARE requirements
Karatekin E, Di Giovanni J, Iborra C, Coleman J, O'Shaughnessy B, Seagar M, Rothman JE. A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proceedings Of The National Academy Of Sciences Of The United States Of America 2010, 107: 3517-3521. PMID: 20133592, PMCID: PMC2840481, DOI: 10.1073/pnas.0914723107.Peer-Reviewed Original Research