Protein-Protein interactions: . 1953: structure of DNA determined. · 2003: complete sequence of human genome. · Human genome ~ 3 billion bps. 20-25,00 encoded proteins. Genome not much larger than some simpler organisms. · Info stored in genes. . Many variants of proteins by variable splicing and post- translational modifications. · Structural genomes: 3D structures of all gene products. · Interaction proteomics: how they interact to mediate functional activities. No. gene products/structure and genes very large. . Molecular interactions occur through aspects of molecular complementarity. Variability in flexible structures/bonds/h.phobicity etc. · Specific molecular recognition events mediate biological processes. · Applications from structural biology: drug design e.g. Glivec which targets a kinase involved in myeloid leukaemia. · IgE interaction networks control allergic and inflammatory immune reactions. · Able to map protein-protein interactions of entire proteomes. · Interactions can be transient or obligate- always together/permanently. · Interaction hubs: data hubs: only 2 at a time, or party hubs/promiscuous proteins: more than 2. How are protein-protein interaction partners identified? · Genetic based approach: - Yeast two- hybrid principle. - Each one of the 25,000 proteins have a genetic construct. Each target is put into a plasmid. - Protein A placed in bait plasmid. - Protein B into prey plasmid and co-transfect into bacterial cell. - Both bait and prey plasmid, take unknown proteins and put as fusion proteins to give proteins that have a defined functionality to them. - If proteins A and B bind to each other, two different fusion proteins are brought into close proximity and proteins have a functionality that result in expression of target gene/phenotypic outcome. - Very binary in character. - Interactions don't distinguish between date and party hubs- two at a time. · Biochemical based approach: Searching for interacting partners: The yeast two-hybrid principle yeast prey-plasmid 4 transformation prey fusionprotein AD interaction AD translocation Into the nucleus nucleus AD BD bait fusionprotein 8-Gal reporter gene transcription and translation bait-plasmid AD activating domain binding domain
- Mass spec. - Co- immunoprecipitations: used antibody to precipitate: place on solid surface. - Cell which contains target protein is slightly disrupted to remove membranes-> purified cytoplasm. - Antibody on a bead mixed in with this and binds to target protein with high specificity. - Get rid of anything not bound to it by centrifugation -> interaction between antibody and protein A and anything else associated. - Elution step: get rid of antibody. - Mixture applied to mass spec to generate sequences and see what the mix of proteins here are. - Pull downs. Co-Immunoprecipitation (Co-IP) as a method for confirming PPIs a Immunochemical purification b One-step affinity purification Protein sequence fused to tag C Two-step affinity purification Protein sequence fused to tandem tag (a) Homologous nocombination (b) Transient tranafuction (c) Retroviral gone translu Expression of tagged proteins (a) Homologous nacombination (b) Transient transfuction (c) Retroviral gana transtur Expression of tagged protein containing two tags spaced by a cleavable linker Endogeneous complex Assembly of complex containing tagged protein Assembly of complex containing