NMR study of a single-stranded DNA binding to the C-terminal
domain of the protease from murine intracisternal A-type
particle

a 2008

NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle

MOTÁČKOVÁ, Veronika, Monika KUBÍČKOVÁ, Martin ŠVEC, Milan KOŽÍŠEK, Lukáš ŽÍDEK et. al.

Basic information

Original name

NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle

Name in Czech

NMR studie retrovirové proteasy se zaměřením na vazbu jednovláknových nukleových kyselin k C-terminální doméně

Name (in English)

NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle

Authors

MOTÁČKOVÁ, Veronika, Monika KUBÍČKOVÁ, Martin ŠVEC, Milan KOŽÍŠEK, Lukáš ŽÍDEK, Jan KONVALINKA and Vladimír SKLENÁŘ

Edition

XXIII International Conference on Magnetic Resonance in Biological Systems San Diego, 2008

Other information

Type of outcome

Konferenční abstrakt

Confidentiality degree

není předmětem státního či obchodního tajemství

Organization unit

Faculty of Science

Abstract

V originále

Murine intracisternal A-type particles are endogenous betaretroviruses which assemble and bud at the membranes of the mouse endoplasmic reticulum where they accumulate. They do not undergo maturation which involves proteolytic processing of viral proteins. Proteases play a crucial role in the retroviral replication cycle but the mechanism of their regulation remains unclear. The C-terminal domain of the retroviral proteases, rich in glycines, has been proposed to bind RNA. Biochemical tests showed that the C-terminal domain binds single-stranded oligonucleotides (both RNA and DNA) without inhibiting the proteolytic activity. The protocols of the overexpression on minimal media have been optimized and C-13, N-15, H-2 labeled full-length protease samples were prepared. A set of triple resonance experiments have been measured. Backbone assignment covering 95 % of the sequence has been achieved. Interactions between the protease and a single-stranded DNA 20-mer have been studied by titrating the protease sample with the DNA oligomer. The titration was monitored in 2D HN-HSQC and 1D proton NMR spectra. Results revealed stoichiometry of 2 protease molecules per oligonucleotide at low DNA concentrations. Signs of aggregation were observed as the DNA concentration increased. Residues mostly effected by the DNA binding were identified in the positively charged fragment between Ala 117 and Lys 127.

Links

LC06030, research and development project

Name: Biomolekulární centrum

Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular centre

MSM0021622413, plan (intention)

Name: Proteiny v metabolismu a při interakci organismů s prostředím

Investor: Ministry of Education, Youth and Sports of the CR, Proteins in metabolism and interaction of organisms with the environment

Citovat

MOTÁČKOVÁ, Veronika, Monika KUBÍČKOVÁ, Martin ŠVEC, Milan KOŽÍŠEK, Lukáš ŽÍDEK, Jan KONVALINKA and Vladimír SKLENÁŘ. NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle. In XXIII International Conference on Magnetic Resonance in Biological Systems San Diego. 2008.

@proceedings{830084,
   author = {Motáčková, Veronika and Kubíčková, Monika and Švec, Martin and Kožíšek, Milan and Žídek, Lukáš and Konvalinka, Jan and Sklenář, Vladimír},
   booktitle = {XXIII International Conference on Magnetic Resonance in Biological Systems San Diego},
   title = {NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle},
   year = {2008}
}

TY  - CONF
ID  - 830084
AU  - Motáčková, Veronika - Kubíčková, Monika - Švec, Martin - Kožíšek, Milan - Žídek, Lukáš - Konvalinka, Jan - Sklenář, Vladimír
PY  - 2008
TI  - NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle
N2  - Murine intracisternal A-type particles are endogenous betaretroviruses which assemble and bud at the membranes of the mouse endoplasmic reticulum where they accumulate. They do not undergo maturation which involves proteolytic processing of viral proteins. Proteases play a crucial role in the retroviral replication cycle but the mechanism of their regulation remains unclear. The C-terminal domain of the retroviral proteases, rich in glycines, has been proposed to bind RNA. Biochemical tests showed that the C-terminal domain binds single-stranded oligonucleotides (both RNA and DNA) without inhibiting the proteolytic activity. The protocols of the overexpression on minimal media have been optimized and C-13, N-15, H-2 labeled full-length protease samples were prepared. A set of triple resonance experiments have been measured. Backbone assignment covering 95 % of the sequence has been achieved. Interactions between the protease and a single-stranded DNA 20-mer have been studied by titrating the protease sample with the DNA oligomer. The titration was monitored in 2D HN-HSQC and 1D proton NMR spectra. Results revealed stoichiometry of 2 protease molecules per oligonucleotide at low DNA concentrations. Signs of aggregation were observed as the DNA concentration increased. Residues mostly effected by the DNA binding were identified in the positively charged fragment between Ala 117 and Lys 127.
ER  -

MOTÁČKOVÁ, Veronika, Monika KUBÍČKOVÁ, Martin ŠVEC, Milan KOŽÍŠEK, Lukáš ŽÍDEK, Jan KONVALINKA and Vladimír SKLENÁŘ. NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle. In \textit{XXIII International Conference on Magnetic Resonance in Biological Systems San Diego}. 2008.

Detailed Information on Publication Record