BS2520 - Protein Structure and Function Lecture 5 Protein-Protein Interactions Protein-Protein Interactions - A specific physical contact with molecular docking between proteins that occur in a cell or living organism in vivo. - The interaction interface should be intentional and not accidental. - The interaction interface should be non-generic. - PPIs are stable and transient. Example of stable PPIs: - Some protein assemblies are stable macromolecular protein complexes such as multi-subunit proteins e.g. glutamate receptors, voltage dependent Kv, ATPase. - Other protein assemblies are only built to carry out transient actions and can form and breakdown transiently. A signal peptide is recognised by signal recognition particle (SRP). SRPs is a multi-protein complex. SRPs interact with SRP receptors. The removal of signal peptide is catalysed by signal peptidase. Signal sequences show no conservation of sequences. Generally: - They begin with 1- amino acids of the N terminus. - They are between 20 and 30 residues in length. - They have a central hydrophobic core. - Flanked on the N-terminal side by a positively charged stretch of polar residues. - C-terminal region is neutral but polar. Vesicles trafficking and targeting is an example of PPI: - Formatting of the vesicles by budding from the membrane of the ER, Golgi and the plasma membrane and selection of their contents is mediated by PPI between cargo receptor, adaptin, clathrin and dynamin. - Vesicle trafficking and targeting. Each vesicle has a specific targeting signal. - Vesicles are moved long distances by cytoskeleton-based motor proteins and short distances by diffusion. - Specific attachment of vesicles to their target membranes and membrane fusion are mediated by SNARE proteins. - Vesicle fusion is mediated by three proteins which are necessary and sufficient to cause vesicle fusion.
Immunoprecipitation This is a popular technique for protein purification and relies on PPI to capture the antigen of interest. An antibody against a specific target antigen forms an immune complex with that target is separated from the rest of unrelated proteins, the captured antigen in then eluted. Co-immunoprecipitation is a popular technique for protein interaction discovery. It relies on PPI to capture the antigen of interest and relies on PPI to identify and/or capture the protein which interacts with the antigen. Pull-down assays for the analysis of PPI: - Pull-down assays are a form of affinity purification. - A bait protein is used instead of an antibody.