NMR spectroscopy allows the study of biomolecules in close-to-native conditions. Structural information can be inferred from the NMR spectra when an assignment is available. Protein assignment is usually a time-consuming task, being specially challenging in the case of large, supramolecular systems. Here, we present an extension of existing state-of-the-art strategies for methyl group assignment that partially overcomes signal overlapping and other difficulties associated to isolated methyl groups. Our approach exploits the ability of proteins to populate two or more conformational states, allowing for unique NOE restraints in each protein conformer. The method is compatible with automated assignment algorithms, granting assignments beyond the limits of a single protein state. The approach also benefits from long-range structural restraints obtained from metal-induced pseudocontact shifts (PCS) and paramagnetic relaxation enhancements (PREs). We illustrate the method with the complete assignment of the 199 methyl groups of a MILproSVproSAT methyl-labeled sample of the UDP-glucose pyrophosphorylase enzyme from Leishmania major (LmUGP). Protozoan parasites of the genus Leishmania causes Leishmaniasis, a neglected disease affecting over 12 million people worldwide. LmUGP is responsible for the de novo biosynthesis of uridine diphosphate-glucose, a precursor in the biosynthesis of the dense surface glycocalyx involved in parasite survival and infectivity. NMR experiments with LmUGP and related enzymes have the potential to unravel new insights in the host resistance mechanisms used by Leishmania major. Our efforts will help in the development of selective and efficient drugs against Leishmania.
[NMR paper] Unambiguous Side-Chain Assignments for Solid-State NMR Structure Elucidation of Nondeuterated Proteins via a Combined 5D/4D Side-Chain-to-Backbone Experiment
Unambiguous Side-Chain Assignments for Solid-State NMR Structure Elucidation of Nondeuterated Proteins via a Combined 5D/4D Side-Chain-to-Backbone Experiment
Owing to fast-magic-angle-spinning technology, proton-detected solid-state NMR has been facilitating the analysis of insoluble, crystalline, sedimented, and membrane proteins. However, potential applications have been largely restricted by limited access to side-chain resonances. The recent availability of spinning frequencies exceeding 100 kHz in principle now allows direct probing of all protons without the need for partial...
[NMR paper] Backbone-independent NMR resonance assignments of methyl probes in large proteins.
Backbone-independent NMR resonance assignments of methyl probes in large proteins.
Related Articles Backbone-independent NMR resonance assignments of methyl probes in large proteins.
Nat Commun. 2021 Jan 29;12(1):691
Authors: Nerli S, De Paula VS, McShan AC, Sgourakis NG
Abstract
Methyl-specific isotope labeling is a powerful tool to study the structure, dynamics and interactions of large proteins and protein complexes by solution-state NMR. However, widespread applications of this methodology have been limited by challenges in...
Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methylâ??methyl NOESY
Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methylâ??methyl NOESY
Abstract
Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate,...
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[NMR paper] Specific (13)C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.
Specific (13)C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.
Specific (13)C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.
J Magn Reson. 2015 Jan 6;252C:10-19
Authors: Fasshuber HK, Demers JP, Chevelkov V, Giller K, Becker S, Lange A
Abstract
Here we present an isotopic labeling strategy to easily obtain unambiguous long-range...
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Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Abstract Methyl 13CHD2 isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from -glucose/D2O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Alaβ, Thrγ2) is possible using simple â??out-and-backâ?? NMR methodology. Such selective methyl-detected â??out-and-backâ?? NMR experiments allow complete assignments of threonine γ2...
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Narrow carbonyl resonances in proton-diluted proteins facilitate NMR assignments in t
Abstract HNCO/HNCACO type correlation experiments are an alternative for assignment of backbone resonances in extensively deuterated proteins in the solid-state, given the fact that line widths on the order of 14â??17 Hz are achieved in the carbonyl dimension without the need of high power decoupling. The achieved resolution demonstrates that MAS solid-state NMR on extensively deuterated proteins is able to compete with solution-state NMR spectroscopy if proteins are investigated with correlation times Ï? c that exceed 25 ns.
Content Type Journal Article
DOI...
Ligand-induced structural transitions combined with paramagnetic ions facilitate unambiguous NMR assignments of methyl groups in large proteins
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