Document text
Principal Investigator: Kallol Gupta
Organization: YALE UNIVERSITY
Fiscal Year: 2024
Award: $351,750
Funding agency: National Institute of General Medical Sciences
Abstract
In the crowded milieu of the membrane, membrane proteins, with other soluble and membrane-associated
proteins, and lipids form a large number of dynamic and transient protein complexes that in turn govern
cellular physiology. There is also mounting evidence that both independent membrane protein-lipid
interactions, as well as bulk biophysical properties of the host membrane often regulate these assemblies.
Hence, to understand how associations between specific membrane proteins help a cell responds to an
external stimulus, we need to study the oligomeric assemblies of the respective proteins directly from the
lipid bilayer environment. This brings us to the primary challenge of studying membrane protein-lipid
interactions. The existing tools to study such interactions lack this critical ability to perform molecular
analysis directly from the bilayer environment. Addressing this challenge, the overarching goal of this
project is to develop a novel experimental platform that enables analysis of MP complexes directly from in
vitro lipid bilayers, which can be customized to a target cellular membrane. To this end, we will combine
lipid vesicle technologies with native mass spectrometry (nativeMS). In Aim 1, taking a set of ten different
standard oligomeric membrane proteins we will develop an experimental method that enables us to
determine their oligomeric states directly from a range of lipid vesicles mimicking different physiological
membranes. We will validate and benchmark our results against the known oligomeric masses of each of
these proteins. This will establish the applicability of our platform to detect a wide range of membrane
proteins from a variety of lipid bilayer environments. In Aim 2, we will develop an experimental strategy that
enables us to directly determine the specifically bound lipid binds and where do they bind. To this end, in
collaboration with Thermo Fisher Scientific, we will combine ECD fragmentation with lipid vesicle nativeMS
platform. Together, upon successful completion, these two Aims will provide an arsenal of new
technologies to study the oligomeric organization of membrane proteins and lipids directly from a
physiologically relevant lipid bilayer. In Aim 3, we will apply this to a complex biological system to address
an outstanding question in neurobiology; how neurotransmitter filled synaptic vesicles attain their ultrafast
speed of fusion. To this end, we will specifically target the role of synaptophysin, a synaptic vesicle
membrane protein which has been linked to various neurological disorders. The experiment proposed can
bring out critical mechanist and structural insight to understand neuronal signal transduction and related
disease-specific impairments. In the long run, impairment of associations between membrane proteins has
been linked to several pathophysiological conditions ranging from neurodegeneration to cancer. We are
confident that the proposed platform will have a transformative role in studying a wide range of biological
processes and associated disease states.
Terms: <Ablation><Accounting><Address><Assay><Benchmarking><Best Practice Analysis><Binding><Binding Sites><Bioassay><Biochemical><Biologic Models><Biological><Biological Assay><Biological Function><Biological Models><Biological Process><Cancers><Cell Body><Cell Communication and Signaling><Cell Function><Cell Membrane Lipids><Cell Physiology><Cell Process><Cell Signaling><Cell membrane><Cells><Cellular Expansion><Cellular Function><Cellular Growth><Cellular Membrane><Cellular Physiology><Cellular Process><Cellular Regulation><Chemicals><Collaborations><Combining Site><Complex><Crowding><Cytoplasmic Membrane><Data><Detection><Detergents><Disease><Disorder><Endoplasmic Reticulum><Environment><Ergastoplasm><Genetic Alteration><Genetic Change><Genetic defect><Goals><Human><Impairment><In Vitro><Intracellular Communication and Signaling><Lateral><Link><Lipid Bilayers><Lipid Binding><Lipids><Liquid substance><Malignant Neoplasms><Malignant Tumor><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Membrane><Membrane Lipids><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Methodology><Methods><Mitochondria><Model System><Modern Man><Molecular><Molecular Analysis><Molecular Interaction><Mutation><NSF attachment protein receptor><Nerve Cells><Nerve Degeneration><Nerve Impulse Transmission><Nerve Transmission><Nerve Transmitter Substances><Nerve Unit><Nervous System Diseases><Nervous System Disorder><Neural Cell><Neurobiology><Neurocyte><Neurologic Disorders><Neurological Disorders><Neuron Degeneration><Neuronal Transmission><Neurons><Neurotransmitters><Pathologic><Physiologic><Physiological><Plasma Membrane><Play><Property><Proteins><Proteome><Protocol><Protocols documentation><Reactive Site><Role><SNAP receptor><SNARE><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Speed><Stimulus><Subcellular Process><Surface Proteins><Synapses><Synaptic><Synaptic Vesicle P38 Membrane Protein><Synaptic Vesicle Protein P38><Synaptic Vesicles><Synaptophysin><System><Technology><Vesicle><axon signaling><axon-glial signaling><axonal signaling><benchmark><biologic><biological signal transduction><biophysical characteristics><biophysical characterization><biophysical measurement><biophysical parameters><biophysical properties><cell growth><cell growth regulation><cellular targeting><complex biological systems><detection platform><detection system><drug market><experiment><experimental research><experimental study><experiments><fluid><genome mutation><glia signaling><glial signaling><insight><lipid bilayer membrane><lipid bound><liquid><malignancy><membrane assembly><membrane reconstitution><membrane structure><mitochondrial><neoplasm/cancer><nerve signaling><neural degeneration><neural signaling><neurobiological><neurodegeneration><neurodegenerative><neurological degeneration><neurological disease><neuronal><neuronal degeneration><neuronal signaling><neurotransmission><neurotransmitter release><new technology><next generation><novel><novel technologies><plasmalemma><pressure><protein complex><reconstitute><reconstitution><social role><soluble N-ethylmaleimide-sensitive-factor attachment protein receptor><stoichiometry><synapse><technological innovation><tool><vesicle release><vesicular release>