15N NMR Spectroscopy in Structural Analysis

The proteasome is able to create spliced Ags, in which two distant parts of a protein are excised and ligated together to form a novel peptide, for presentation by MHC class I molecules. These noncontiguous epitopes are generated via a transpeptidation reaction catalyzed by the proteasomal active sites. Transpeptidation reactions in the proteasome follow explicit rules and occur particularly efficiently when the C-terminal ligation partner contains a lysine or arginine residue at the site of ligation. Lysine contains two amino groups that theoretically may both participate in ligation reactions, implying that potentially not only peptide but also isopeptide linkages could be formed. Using nuclear magnetic resonance spectroscopy, we demonstrate in the present study that the proteasome can use the ε-amino group of an N-terminal lysine residue in transpeptidation reactions to create a novel type of posttranslationally modified epitopes. We show that the overall efficiency of ε ligation is only 10-fold lower as compared with α ligation, suggesting that the proteasome can produce sufficient isopeptide Ag to evoke a T cell response. Additionally, we show that isopeptides are more stable toward further proteasomal processing than are normal peptides, and we demonstrate that isopeptides can bind to HLA-A2.1 and HLA-A3 with high affinity. These properties likely increase the fraction of ε-ligated peptides presented on the cell surface for CD8+ T cell surveillance. Finally, we show that isopeptide Ags are immunogenic in vivo. We postulate that ε ligation is a genuine posttranslational modification, suggesting that the proteasome can create a novel type of Ag that is likely to play a role in immunity.

show abstract

“…1C). As direct detection of 15 N atoms suffers from low sensitivity and long relaxation times (30), 15 N Fig. 2A) (31).…”

Section: « Ligation Can Compete With a Ligation In Proteasomal Ligatimentioning

confidence: 99%

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage

Berkers

Jong

Schuurman

et al. 2015

The Journal of Immunology

View full text Add to dashboard Cite

show abstract

“…In coordination chemistry with nitrogen-donor ligands, 15 N NMR spectroscopy has been used as a probe to assign the coordination in solution. [31][32][33] The nitrogen atom in such ligands is exposed to the most dramatic changes upon coordination, but also the nitrogen chemical shifts are highly sensitive to structural changes, more so than 13 C chemical shifts. [34] Although the low natural abundance of 15 N and its low gyromagnetic ratio make direct observation of the 15 N nuclide difficult, this has been largely circumvented by the advent of the inverse-detected long-range heteronuclear shift correlation techniques, introduced by Bax and Summers.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Synthesis and NMR Analysis of 1,4‐Disubstituted 1,2,3‐Triazoles Tethered to Pyridine, Pyrimidine, and Pyrazine Rings

2014

View full text Add to dashboard Cite

A combinatorial modular approach based on the “click” copper(I)‐catalysed azide–alkyne cycloaddition reaction was used to prepare a library of selected 1,4‐disubstituted 1,2,3‐triazoles differently functionalized with heteroaryl groups including pyridine, pyrimidine, and pyrazine. Three different copper(I) sources, i.e., CuSO4/sodium ascorbate, CuBr(PPh3)3, and (CuOTf)2·C6H6 were used to promote the coupling reactions of a range of aryl, heteroaryl, and heteroarylmethyl azides with alkyne “click” partners. As the target heteroaryl triazoles were designed to serve as advanced ligands or ligand precursors for metal coordination, they were fully characterized by 1H, 13C, and 15N NMR spectroscopy. The resonances were assigned on the basis of 1D and 2D NMR experiments. 1H–15N gs‐HMBC was used as a practical tool to determine 15N NMR chemical shifts at the natural abundance level of the 15N isotope.

show abstract

“…Better experimental access is available to long-range 1 19 Long-range 1 H-15 N 2D NMR experiments at natural abundance, which might also be of additional help in the assembly of substructural fragments and the elucidation of the structure, are also now possible and the subject of a recent review. 20,21 The second structure shown in Fig. 7 is assigned as the structure.…”

Section: Discussionmentioning

confidence: 99%

Quindolinocryptotackieine: the elucidation of a novel indoloquinoline alkaloid structure through the use of computer‐assisted structure elucidation and 2D NMR

Blinov¹,

Elyashberg²,

Martirosian³

et al. 2003

Magnetic Reson in Chemistry

View full text Add to dashboard Cite

Numerous indoloquinoline alkaloid structures have been identified from extracts of the West African plant Cryptolepis sanguinolenta. Recently, through the use of 2D NMR methods and cryogenic NMR probe technology in conjunction with computer-assisted structure elucidation (CASE) methods, the structures of some chemical degradation products of this family of alkaloids have also been reported. We now report the characterization of a novel indoloquinoline dimeric alkaloid, quindolinocryptotackieine, through the extensive utilization of CASE methods. The NMR data presented here were collected over a decade earlier before the elucidation of the structure was possible, since manual analysis did not present a conclusive structure, whereas CASE produced a series of structures from which the structure could be verified. The original mass spectrometric (MS) data collected for the sample were problematic. Contemporary MS data were instead recollected from remaining small quantities of this alkaloid using modern instrumentation. The re-collected data gave a usable molecular ion and several key fragment ions that were diagnostically useful.

show abstract

15N NMR Spectroscopy in Structural Analysis

Cited by 182 publications

References 4 publications

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage

Synthesis and NMR Analysis of 1,4‐Disubstituted 1,2,3‐Triazoles Tethered to Pyridine, Pyrimidine, and Pyrazine Rings

Quindolinocryptotackieine: the elucidation of a novel indoloquinoline alkaloid structure through the use of computer‐assisted structure elucidation and 2D NMR

Contact Info

Product

Resources

About