Selective isotope labeling for NMR structure determination of
proteins in complex with unlabeled ligands

J 2019

Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands

TRIPSIANES, Konstantinos, U. SCHUTZ, L. EMMANOUILIDIS, G. GEMMECKER, M. SATTLER et. al.

Základní údaje

Originální název

Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands

Autoři

TRIPSIANES, Konstantinos (300 Řecko, garant, domácí), U. SCHUTZ (276 Německo), L. EMMANOUILIDIS (276 Německo), G. GEMMECKER (276 Německo) a M. SATTLER (276 Německo)

Vydání

Journal of biomolecular NMR, Dordrecht, Springer, 2019, 0925-2738

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Nizozemské království

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 2.634

Kód RIV

RIV/00216224:14740/19:00110453

Organizační jednotka

Středoevropský technologický institut

DOI

http://dx.doi.org/10.1007/s10858-019-00241-9

UT WoS

000468272700007

Klíčová slova anglicky

NMR spectroscopy; Isotope labeling; Protein-ligand interactions; NOE

Štítky

rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 8. 3. 2020 19:26, Mgr. Pavla Foltynová, Ph.D.

Anotace

V originále

The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific C-13, N-15 and H-2 isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.

Návaznosti

LQ1601, projekt VaV

Název: CEITEC 2020 (Akronym: CEITEC2020)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, CEITEC 2020

Citovat

TRIPSIANES, Konstantinos, U. SCHUTZ, L. EMMANOUILIDIS, G. GEMMECKER a M. SATTLER. Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands. Journal of biomolecular NMR. Dordrecht: Springer, 2019, roč. 73, 3-4, s. 183-189. ISSN 0925-2738. Dostupné z: https://dx.doi.org/10.1007/s10858-019-00241-9.

@article{1549789,
   author = {Tripsianes, Konstantinos and Schutz, U. and Emmanouilidis, L. and Gemmecker, G. and Sattler, M.},
   article_location = {Dordrecht},
   article_number = {3-4},
   doi = {http://dx.doi.org/10.1007/s10858-019-00241-9},
   keywords = {NMR spectroscopy; Isotope labeling; Protein-ligand interactions; NOE},
   language = {eng},
   issn = {0925-2738},
   journal = {Journal of biomolecular NMR},
   title = {Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands},
   url = {https://link.springer.com/content/pdf/10.1007%2Fs10858-019-00241-9.pdf},
   volume = {73},
   year = {2019}
}

TY  - JOUR
ID  - 1549789
AU  - Tripsianes, Konstantinos - Schutz, U. - Emmanouilidis, L. - Gemmecker, G. - Sattler, M.
PY  - 2019
TI  - Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands
JF  - Journal of biomolecular NMR
VL  - 73
IS  - 3-4
SP  - 183-189
EP  - 183-189
PB  - Springer
SN  - 09252738
KW  - NMR spectroscopy
KW  - Isotope labeling
KW  - Protein-ligand interactions
KW  - NOE
UR  - https://link.springer.com/content/pdf/10.1007%2Fs10858-019-00241-9.pdf
L2  - https://link.springer.com/content/pdf/10.1007%2Fs10858-019-00241-9.pdf
N2  - The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific C-13, N-15 and H-2 isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.
ER  -

TRIPSIANES, Konstantinos, U. SCHUTZ, L. EMMANOUILIDIS, G. GEMMECKER a M. SATTLER. Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands. \textit{Journal of biomolecular NMR}. Dordrecht: Springer, 2019, roč.~73, 3-4, s.~183-189. ISSN~0925-2738. Dostupné z: https://dx.doi.org/10.1007/s10858-019-00241-9.

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