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.
An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins
An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins
Abstract
Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded...
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01-04-2016 07:49 PM
Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags
Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags
Abstract
Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Î?Ď?) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative...
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11-23-2015 06:58 PM
[NMR paper] CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
Related Articles CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.
J Biomol NMR. 2015 Nov 13;
Authors: Kerfah R, Hamelin O, Boisbouvier J, Marion D
Abstract
A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic...
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11-15-2015 07:55 PM
CH 3 -specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample
CH 3 -specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample
Abstract
A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back 13Câ??13C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu)...
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11-14-2015 03:37 PM
Long-lived nuclear spin states in methyl groups and quantum-rotor-induced polarization
From The DNP-NMR Blog:
Long-lived nuclear spin states in methyl groups and quantum-rotor-induced polarization
Meier, B., et al., Long-lived nuclear spin states in methyl groups and quantum-rotor-induced polarization. J Am Chem Soc, 2013. 135(50): p. 18746-9.
http://www.ncbi.nlm.nih.gov/pubmed/24252212
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02-13-2014 01:42 AM
[NMR paper] Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.
Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--production.springer.de-OnlineResources-Logos-springerlink.gif Related Articles Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.
J Biomol NMR. 2013 Sep 28;
Authors: Mas G, Crublet E, Hamelin O, Gans P, Boisbouvier J
Abstract
The specific protonation of valine and leucine methyl...
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10-01-2013 11:15 PM
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Abstract Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic...
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09-26-2011 06:42 AM
A DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.
A DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.
A DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.
Bioconjug Chem. 2011 Aug 31;
Authors: Graham B, Loh CT, Swarbrick JD, Ung P, Shin J, Yagi H, Jia X, Chhabra S, Barlow N, Pintacuda G, Huber T, Otting G
Abstract
Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to...
Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts
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