NMR as a tool for studies of (partially) disordered proteins and their interactions

PAPOUŠKOVÁ, Veronika, Lukáš ŽÍDEK, Vladimír SKLENÁŘ, Libor KRÁSNÝ and Wiktor KOZMINSKI. NMR as a tool for studies of (partially) disordered proteins and their interactions. In Molecular Perspectives on Protein-Protein Interactions. 2010.

Basic information

• Original name: NMR as a tool for studies of (partially) disordered proteins and their interactions
• Name in Czech: NMR jako nástroj pro studium (částečně) neuspořádaných proteinů a jejich interakcí
• Name (in English): NMR as a tool for studies of (partially) disordered proteins and their interactions
• Authors: PAPOUŠKOVÁ, Veronika, Lukáš ŽÍDEK, Vladimír SKLENÁŘ, Libor KRÁSNÝ and Wiktor KOZMINSKI.
• Edition: Molecular Perspectives on Protein-Protein Interactions, 2010.

Other information

• Type of outcome: Conference abstract
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL
• Organization unit: Faculty of Science

Abstract

One third of eukaryotic proteins, including human, contains disordered regions longer than 30 amino acids. The intrinsically disordered proteins have well-defined biological functions, some of them may adopt a regular structure on binding to their interacting partners, and many of them are related to human diseases. The lack of a unique structure makes the disordered proteins poor targets for crystallographic studies. Therefore, nuclear magnetic resonance (NMR) plays a crucial role in studies of disordered proteins. NMR can provide valuable information on residual secondary structure, possible long-range contacts, internal dynamics of the disordered polypeptide chain, and its structuring upon interactions with other proteins. The first step in NMR investigation of a disordered protein and its interactions is the assignment of observed spectral frequencies to individual atoms in the polypeptide chain. Combination of the structural disorder with a high incidence of sequential repeats often results in spectra with severely overlapped peaks, impossible to assign by the traditional approach. A novel assignment strategy, based on NMR experiments with extreme resolution will be presented. Two examples of partially disordered proteins, involved in protein-protein and/or protein-DNA interactions will be discussed: Retroviral protease of the murine intracisternal A-type particle (assigned by the traditional approach) and delta subunit of RNA polymerase from Bacillus subtillis (assigned by the novel strategy). Results of studies of both proteins interacting with their molecular partners will be presented. The retroviral protease, studied by NMR titration experiment, represents an example of interactions of two unstructured molecules, a disordered protein domain and ss-DNA oligonucleotide. The interactions of the RNA polymerase subunit were studied by gel-shift assay and the interaction surface of the protein, structure of which was determined within the project, was identified by evolutionary analysis. Analysis of internal motions of the subunit, based on NMR relaxation dispersion measurements, showed a strong correlation between conformational changes and intermolecular contacts.

Abstract (in English)

One third of eukaryotic proteins, including human, contains disordered regions longer than 30 amino acids. The intrinsically disordered proteins have well-defined biological functions, some of them may adopt a regular structure on binding to their interacting partners, and many of them are related to human diseases. The lack of a unique structure makes the disordered proteins poor targets for crystallographic studies. Therefore, nuclear magnetic resonance (NMR) plays a crucial role in studies of disordered proteins. NMR can provide valuable information on residual secondary structure, possible long-range contacts, internal dynamics of the disordered polypeptide chain, and its structuring upon interactions with other proteins. The first step in NMR investigation of a disordered protein and its interactions is the assignment of observed spectral frequencies to individual atoms in the polypeptide chain. Combination of the structural disorder with a high incidence of sequential repeats often results in spectra with severely overlapped peaks, impossible to assign by the traditional approach. A novel assignment strategy, based on NMR experiments with extreme resolution will be presented. Two examples of partially disordered proteins, involved in protein-protein and/or protein-DNA interactions will be discussed: Retroviral protease of the murine intracisternal A-type particle (assigned by the traditional approach) and delta subunit of RNA polymerase from Bacillus subtillis (assigned by the novel strategy). Results of studies of both proteins interacting with their molecular partners will be presented. The retroviral protease, studied by NMR titration experiment, represents an example of interactions of two unstructured molecules, a disordered protein domain and ss-DNA oligonucleotide. The interactions of the RNA polymerase subunit were studied by gel-shift assay and the interaction surface of the protein, structure of which was determined within the project, was identified by evolutionary analysis. Analysis of internal motions of the subunit, based on NMR relaxation dispersion measurements, showed a strong correlation between conformational changes and intermolecular contacts.

Links

• GA204/09/0583, research and development project: Name: Delta podjednotka RNA polymerázy z Gram pozitivních bakterií (Acronym: DELTA)

Investor: Czech Science Foundation

• GD301/09/H004, research and development project: Name: Molekulární a strukturní biologie vybraných cytostatik. Od mechanistických studií k chemoterapii rakoviny

Investor: Czech Science Foundation

• 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