1994 — 2002 |
Shin, Yeon-Kyun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Mechanisms of Transmembrane Signaling @ University of California Berkeley
The long term goal of this project is to elucidate the mechanisms of (1) transmembrane molecular switching during signal transduction and (2) the protein transport across membranes. The proposed research explores a new electron paramagnetic resonance (EPR) approach to the investigation of the structure and dynamics of proteins. The strategy is to site-specifically place a nitroxide spin label at any selected site in a protein either using site-directed mutagenesis to replace a native residue with cysteine that provides unique labeling sites for the nitroxide spin labels, or using the synthetic method in which an unnatural amino acid with a nitroxide moiety is directly incorporated. Information on the structure and topology of a protein is obtained from EPR analysis of spin labeled mutants. Association equilibria of membrane receptors can be also studied. Moreover, time-resolved EPR spectroscopy makes it possible to resolve the sequential mechanism of the structural dynamics of spin labeled mutants. With these methods we (i) characterize protein conformational changes; (ii) identify all intermediates; (iii) interpret the kinetics in terms of structure. This unique EPR approach will be applied to two systems: (i) the intact bacterial chemotaxis receptor, a homodimeric membrane protein that is directly involved in signal transduction, for which motion of structural units during transmembrane signaling will be, characterized, defined, and time resolved; (ii) a mitochondrial signal peptide, for which the mechanism of transmembrane potential-dependent translocation will be determined to probe the biophysical principles of protein transport.
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2003 — 2006 |
Shin, Yeon-Kyun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism of Synaptic Snare Assembly
DESCRIPTION (provided by applicant): Membrane fusion is required for neurotransmitter release at the nerve terminal. Formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex bridges the neurotransmitter-loaded synaptic vesicles to the presynaptic plasma membrane, facilitating membrane fusion. The long-term goal of this project is to elucidate the molecular mechanism of SNARE assembly and SNARE-induced membrane fusion. SNARE proteins are amphipathic integral membrane proteins that are not currently amenable to x-ray crystallography and NMR. The present project uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), an established technique for the investigation of structures and topologies of membrane proteins. On the basis of various EPR results a structural model of the protein in the native-like phospholipid bilayer is generated at backbone resolution. In this project, structures and membrane topologies of individual full-length SNARE proteins, their assembly intermediates, and the final complex are determined using SDSL EPR. In particular, emphasis is placed on the domains at the membrane-water interface and the transmembrane domains that are directly involved in driving lipid mixing and fusion pore formation. Amino acid sequences of neuronal SNAREs are similar to those of other SNAREs involved in the endocytic and secretory pathways. Further, the structure of the soluble SNARE core is astonishingly similar to those of viral fusion proteins such as influenza hemagglutinin and HIV gp41. Therefore, what is learned from neuronal SNAREs will have strong implications for other membrane fusion systems. Dysfunction of SNARE assembly results in severe mental illness. Thus, this study will help understand the molecular mechanism of SNARE-related mental illness.
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2003 — 2010 |
Shin, Yeon-Kyun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Yeast Snare Assembly and Membrane Fusion
[unreadable] DESCRIPTION (provided by applicant): Membrane fusion is required for important biological processes such as neurotransmitter release at the synapse and secretion of insulin from the pancreatic p cells. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are the core constituents of the intracellular fusion machinery which is highly conserved from yeast to human. Formation of the SNARE complex between vesicle-associated (v-) SNARE and target membrane (t-) SNARE bridges two membranes, facilitating fusion. The long-term goal of this project is to elucidate the molecular mechanism by which SNAREs mediate membrane fusion. The present project uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), an established technique for the investigation of structures and topologies of membrane proteins. On the basis of various EPR results, a structural model of the protein in the native-like phospholipid bilayer is generated at the backbone resolution. In this project, structures and membrane topologies of individual full-length SNARE proteins and the final complex are determined using SDSL EPR. In particular, emphasis is placed on the transmembrane domains that are directly involved in driving lipid mixing and fusion pore formation. SNARE-mediated membrane fusion may involve multiple intermediates. The newly developed single membrane fusion assay based on total internal reflection (TIRF) microscopy makes it possible to dissect and characterize individual intermediate steps along the fusion pathway in unprecedented detail. In this project, the full dynamics of lipid mixing during fusion will be monitored in real time using this new generation fusion assay. Our studies will be focused on the SNARE system involved in trafficking from Golgi to the plasma membrane in yeast. Amino acid sequences of SNAREs implicated in various endocytic and secretory pathways are very similar to one another. Therefore, what is learned from yeast SNAREs will have general implications. Dysfunction of SNARE assembly and membrane fusion results in severe mental illness and diabetes. Thus, this study will help us to understand the molecular mechanism of SNARE-related illnesses. [unreadable] [unreadable] [unreadable]
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2008 — 2011 |
Shin, Yeon-Kyun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulations of Snare-Dependent Membrane Fusion
[unreadable] DESCRIPTION (provided by applicant): Ca++-triggered synchronized release of the neurotransmitter at the synapse, which underlies neuronal communication and synaptic plasticity, requires membrane fusion. This remarkable process is controlled by an exquisitely orchestrated array of protein-protein interactions. SNARE (soluble N- ethylmaleimide-sensitive factor attachment protein receptor) assembly is the central event that may drive membrane fusion. A Ca++-sensor synaptotagmin I (SytI) and complexins impinge upon SNARE assembly to control the timing and the size of the release. The present project uses the newly developed single fusion assay based on total internal reflection (TIRF) microscopy to investigate the mechanism by which SNARE-dependent fusion is regulated by SytI, complexins, and Ca++. The single fusion assay makes it possible to dissect and characterize individual fusion intermediates in unprecedented detail. Further, this technique allows us to track the dynamic transitions in a single fusion event with msec time resolution. With this powerful new fusion assay, we will dissect how the fusion regulators modulate the individual fusion steps. Further, the present project uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), an established technique for the investigation of structures and topologies of membrane proteins. On the basis of various EPR results, a structural model of the protein in the native- like phospholipid bilayer is generated at the backbone resolution. In this project, conformational changes of SNARE complexes induced by SytI, complexins, and Ca++ are determined using SDSL EPR to gain insights into the structural basis of the synchronized release. The combined approach employing the single fusion assay and SDSL EPR will provide insights into mechanism whereby the synchronized release is manufactured in the neuron. Dysfunction of synchronous neurotransmitter release is linked to hideous mental illnesses such as schizophrenia, autism, and epilepsy, as well as to less serious illnesses including behavioral disorders. The outcomes of the proposed research will lead to the understanding of the mechanism whereby the synchronous release is controlled, which will eventually lead to the mechanism-based design of drugs for metal illnesses. [unreadable] [unreadable] [unreadable]
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2012 — 2015 |
Shin, Yeon-Kyun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Snare-Dependent Vesicle Fusion
The neuron has a remarkable ability to release quanta of neurotransmitters in less than a millisecond in response to the pulse of Ca2+. This fast synchronized release constitutes the fundamental basis of major brain activities. The release requires vesicle fusion, which is orchestrated by the fusion machine SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, a priming agent Complexin (Cpx), and the Ca2+ switch Synatotagmin 1 (Syt1). This research project uses the innovative single-vesicle fusion assay to investigate the mechanism by which SNARE-dependent vesicle fusion is regulated by Cpx, Syt1, and Ca2+. The single-vesicle fusion assay makes it possible to dissect the sequential intermediate stages of vesicle fusion, overcoming major drawbacks of conventional bulk fusion assays. The technique also allows us to track the dynamic transitions in a single fusion event with millisecond time resolution. With this powerful approach, we intend to reconstitute synchronized vesicle fusion in a test tube. Specifically, we test a mechanistic model proposing that Cpx arrests an intermediate stage called hemifusion and that Syt1 delivers the final blow to the fusion machinery at the Ca2+ spike. In parallel, this research program uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), a powerful technique for the investigation of the protein structure at the membrane interface. With EPR we intend to comprehend the structural basis for the regulation of SNARE-dependent fusion necessary for synchronization. The combined approach of the single fusion assay and SDSL EPR will provide important insights into the mechanism by which the synchronized fast release is choreographed in the neuron.
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2016 — 2019 |
Shin, Yeon-Kyun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Snare-Dependent Synaptic Vesicle Fusion
? DESCRIPTION (provided by applicant): The neuron is remarkable in that upon the influx of Ca2+ it synchronizes vesicle fusion, and releases many quanta of neurotransmitters to the synaptic cleft in less than 1 msec. The fast synchronization is orchestrated by interactions between the core fusion machinery SNAREs and auxiliary proteins including a major Ca2+-sensor synaptotagmin 1 (Syt1) and a clamping factor complexin (Cpx). Because release underlies cognition and behavior, toxic agents that undermine the release of neurotransmitter might lead to the symptoms of neurodegenerative diseases such as Parkinson's and Alzheimer's. In this project we use innovative approaches to investigate the mechanism whereby the fusion machinery achieves the synchronization of vesicle fusion. To mimic the native membrane environment we prepare a nanodisc sandwich that harbors a single trans SNARE complex in the middle. Single molecule (sm)FRET and site-directed spin labeling (SDSL) EPR are used to characterize the interactions of SNAREs with auxiliary proteins in the chasm of two nanodiscs. In addition, the drastically improved single vesicle-vesicle fusion assay that can resolve docking, lipid mixing, fusion pore opening, and pore expansion steps, is used to delineate the intervention of regulatory factors onto individual fusion steps. Taken altogether, a comprehensive picture of how synchronization of vesicle fusion is choreographed by the interactions among individual components of the fusion machinery would emerge. For neurodegenerative diseases such as Parkinson's there is an emerging theme of pathophysiology that toxic misfolded oligomers are tampering with the vesicle fusion machinery, leading to disease symptoms. We use EPR to investigate the interaction between ?-synuclein and vesicle (v-)SNARE VAMP2 that takes place on the membrane surface. The outcomes of these investigations are expected to reveal new therapeutic targets for treating symptoms of the Parkinson's disease.
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