Carbon-detected Experiments for Assignment of Proline Rich
Proteins

a 2010

Carbon-detected Experiments for Assignment of Proline Rich Proteins

FIALA, Radovan, Michaela HORNIČÁKOVÁ, Lukáš ŽÍDEK a Norbert MÜLLER

Základní údaje

Originální název

Carbon-detected Experiments for Assignment of Proline Rich Proteins

Název česky

Uhlíkem detekované experimenty pro přiřazení proteinů bohatých na prolin

Autoři

FIALA, Radovan, Michaela HORNIČÁKOVÁ, Lukáš ŽÍDEK a Norbert MÜLLER

Vydání

25th NMR Valtice - Central European NMR Meeting, 2010

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10600 1.6 Biological sciences

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Organizační jednotka

Přírodovědecká fakulta

ISBN

978-80-86441-42-9

Klíčová slova česky

NMR spektroskopie, proteiny, struktura, prolin

Klíčová slova anglicky

NMR spectroscopy, protein, structure, proline

Příznaky

Mezinárodní význam

Změněno: 4. 10. 2010 15:39, doc. RNDr. Radovan Fiala, CSc.

Anotace

ORIG CZ

V originále

The determination of 3D structures of proteins by NMR requires a complete assignment of resonances. For that purpose, suites of experiments allowing both sequential and side-chain assignments have been developed. Standard heteronuclear NMR techniques for the backbone assignment such as HNCA, CBCA(CO)NH, and CBCANH experiments rely on correlating the HN chemical shift of each residue with the C(alpha) and C(beta) chemical shifts of the same and preceding residues. Because proline lacks the HN, these experiments cannot be used to sequentially connect prolines to the preceding residues or to assign the stretches of prolines. Therefore, proline assignment has to be based either on the observation of NOEs to neighboring residues or on the detection of H(alpha) resonances. As the H(alpha) proton resonances often fall close to the resonance of water, their detection often fails. Both the problem of the missing amide proton and the interference of the water signal can be solved using carbon-detection. An extensive set of "protonless" experiments have been proposed for the assignment of fully deuterated proteins and protein complexes with paramagnetic ions. We have applied (H)CbCaCON, (H)CbCaNCO and CON experiments to assign the backbone of proline stretches in the 16 kDa PsbQ protein, which is a part of Photosystem II. Though all detected nuclei are 13C and 15N, the (H)CbCaCON and (H)CbCaNCO start from 1H polarization to increase the sensitivity. The CON experiment was performed also in a proline-selective manner to reduce the crowding of the spectrum and to help to identify the proline residues. With the carbon-detected experiments, we were able to assign 12 out of 13 proline residues in the PsbQ protein using the sample concentration of about 0.3 mM. The experiments were performed on a 600 MHz spectrometer equipped with a TCI cryoprobe.

Česky

Byla navržena metoda přiřazení signálů prolinu v NMR spektrech proteinu založená na přímé detekci uhlíku C-13.

Návaznosti

LC06030, projekt VaV

Název: Biomolekulární centrum

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Biomolekulární centrum

MSM0021622413, záměr

Název: Proteiny v metabolismu a při interakci organismů s prostředím

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Proteiny v metabolismu a při interakci organismů s prostředím

Citovat

FIALA, Radovan, Michaela HORNIČÁKOVÁ, Lukáš ŽÍDEK a Norbert MÜLLER. Carbon-detected Experiments for Assignment of Proline Rich Proteins. In 25th NMR Valtice - Central European NMR Meeting. 2010. ISBN 978-80-86441-42-9.

@proceedings{901013,
   author = {Fiala, Radovan and Horničáková, Michaela and Žídek, Lukáš and Müller, Norbert},
   booktitle = {25th NMR Valtice - Central European NMR Meeting},
   keywords = {NMR spectroscopy, protein, structure, proline},
   language = {eng},
   isbn = {978-80-86441-42-9},
   title = {Carbon-detected Experiments for Assignment of Proline Rich Proteins},
   url = {http://ncbr.chemi.muni.cz/nmrvaltice/},
   year = {2010}
}

TY  - CONF
ID  - 901013
AU  - Fiala, Radovan - Horničáková, Michaela - Žídek, Lukáš - Müller, Norbert
PY  - 2010
TI  - Carbon-detected Experiments for Assignment of Proline Rich Proteins
SN  - 9788086441429
KW  - NMR spectroscopy, protein, structure, proline
UR  - http://ncbr.chemi.muni.cz/nmrvaltice/
N2  - The determination of 3D structures of proteins by NMR requires a complete assignment of resonances. For that purpose, suites of experiments allowing both sequential and side-chain assignments have been developed. Standard heteronuclear NMR techniques for the backbone assignment such as HNCA, CBCA(CO)NH, and CBCANH experiments rely on correlating the HN chemical shift of each residue with the C(alpha) and C(beta) chemical shifts of the same and preceding residues. Because proline lacks the HN, these experiments cannot be used to sequentially connect prolines to the preceding residues or to assign the stretches of prolines. Therefore, proline assignment has to be based either on the observation of NOEs to neighboring residues or on the detection of H(alpha) resonances. As the H(alpha) proton resonances often fall close to the resonance of water, their detection often fails. Both the problem of the missing amide proton and the interference of the water signal can be solved using carbon-detection. An extensive set of "protonless" experiments have been proposed for the assignment of fully deuterated proteins and protein complexes with paramagnetic ions. We have applied (H)CbCaCON, (H)CbCaNCO and CON experiments to assign the backbone of proline stretches in the 16 kDa PsbQ protein, which is a part of Photosystem II. Though all detected nuclei are 13C and 15N, the (H)CbCaCON and (H)CbCaNCO start from 1H polarization to increase the sensitivity. The CON experiment was performed also in a proline-selective manner to reduce the crowding of the spectrum and to help to identify the proline residues. With the carbon-detected experiments, we were able to assign 12 out of 13 proline residues in the PsbQ protein using the sample concentration of about 0.3 mM. The experiments were performed on a 600 MHz spectrometer equipped with a TCI cryoprobe.
ER  -

FIALA, Radovan, Michaela HORNIČÁKOVÁ, Lukáš ŽÍDEK a Norbert MÜLLER. Carbon-detected Experiments for Assignment of Proline Rich Proteins. In \textit{25th NMR Valtice - Central European NMR Meeting}. 2010. ISBN~978-80-86441-42-9.

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