title: Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts.
abstract: Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca2+-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA.|Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA.|Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Yun-Zu
authors.initials: YZ
authors.lastname: Pan
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA.|Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA.|Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Bradley
authors.initials: B
authors.lastname: Quade
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA.|Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA.|Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Kyle D
authors.initials: KD
authors.lastname: Brewer
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Monika
authors.initials: M
authors.lastname: Szabo
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: James D
authors.initials: JD
authors.lastname: Swarbrick
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Bim
authors.initials: B
authors.lastname: Graham
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA. Jose.Rizo-Rey@UTSouthwestern.edu.|Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA. Jose.Rizo-Rey@UTSouthwestern.edu.|Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Jose.Rizo-Rey@UTSouthwestern.edu.
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
mesh_terms: D000818:Animals; D007553:Isotope Labeling; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D028581:Lanthanoid Series Elements; D046912:Multiprotein Complexes; D050600:SNARE Proteins
keywords: Membrane fusion; Methyl assignment; Neurotransmitter release; Pseudocontact shifts; SNAREs