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.
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.
Pajunen
Maria
M
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland.
Turakainen
Hilkka
H
Poussu
Eini
E
Peränen
Johan
J
Vihinen
Mauno
M
Savilahti
Harri
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D015723:Gene Library; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism
D008565:Membrane Proteins; C431146:SYTL1 protein, human; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins
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