High-precision mapping of protein-protein interfaces

an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform

Maria Pajunen, Hilkka Turakainen, Eini Poussu, Johan Peränen, Mauno Vihinen, Harri Savilahti

    Research output: Contribution to journalArticleScientificpeer-review

    Abstract

    Understanding networks of protein-protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein-protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1.
    Original languageEnglish
    JournalNucleic Acids Research
    Volume35
    Issue number16, e103, [11] s
    ISSN0305-1048
    DOIs
    Publication statusPublished - 2007
    MoE publication typeA1 Journal article-refereed

    Cite this

    @article{5a5efd0e37eb48f7a390fec1ddc19a8c,
    title = "High-precision mapping of protein-protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform",
    abstract = "Understanding networks of protein-protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein-protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1.",
    author = "Maria Pajunen and Hilkka Turakainen and Eini Poussu and Johan Per{\"a}nen and Mauno Vihinen and Harri Savilahti",
    year = "2007",
    doi = "10.1093/nar/gkm563",
    language = "English",
    volume = "35",
    journal = "Nucleic Acids Research",
    issn = "0305-1048",
    publisher = "Oxford University Press",
    number = "16, e103, [11] s",

    }

    High-precision mapping of protein-protein interfaces : an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform. / Pajunen, Maria; Turakainen, Hilkka; Poussu, Eini; Peränen, Johan; Vihinen, Mauno; Savilahti, Harri.

    In: Nucleic Acids Research, Vol. 35, No. 16, e103, [11] s, 2007.

    Research output: Contribution to journalArticleScientificpeer-review

    TY - JOUR

    T1 - High-precision mapping of protein-protein interfaces

    T2 - an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform

    AU - Pajunen, Maria

    AU - Turakainen, Hilkka

    AU - Poussu, Eini

    AU - Peränen, Johan

    AU - Vihinen, Mauno

    AU - Savilahti, Harri

    PY - 2007

    Y1 - 2007

    N2 - Understanding networks of protein-protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein-protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1.

    AB - Understanding networks of protein-protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein-protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1.

    U2 - 10.1093/nar/gkm563

    DO - 10.1093/nar/gkm563

    M3 - Article

    VL - 35

    JO - Nucleic Acids Research

    JF - Nucleic Acids Research

    SN - 0305-1048

    IS - 16, e103, [11] s

    ER -