Protein-Protein Interactions Lecture 2 PDF

“Protein-protein interactions” Dr. Manal Abouelwafa Badr University in Assiut Biotechnology Department [email protected] This course will give an introduction to the field of proteomic and Genomics and t...

“Protein-protein interactions” Dr. Manal Abouelwafa Badr University in Assiut Biotechnology Department [email protected] This course will give an introduction to the field of proteomic and Genomics and the available proteomic technologies and Protein-protein interactions tools. Lecture 2 14 Oct, 2024.  Co-immunopercipitation Contents 2 hybrid assay Yeast two-hybrid assay Far western blot Protein microarry Fluorescence resonance energy transfer (FRET) Protein microarray Confocal microscopy Introduction Protein-protein interactions Important field in cell biology, biochemistry Localization and trafficking posttranslational modifications signaling networks Also important field in viral replication Very difficult to predict Two main patterns: domain-domain interactions domain-peptide interactions  Protein-Protein Interaction Proteins work together by actually binding to form multicomponent complexes that carry out specific functions. These functional units can be as simple as dimeric transcription-factor complexes or as complex as the 30-plus component systems that form ribosomes. Biochemists believe that all proteins bind to or interact with at least one other protein. The discovery that proteins in higher organisms (e.g., human and mouse) contain higher numbers of functional domains suggests that many of these proteins have multiple associations. Understanding how protein complexes work is essential to understanding how cells work as systems. Identifying Protein-Protein Interaction In the pregenomic era, immunoprecipitation was the primary means of determining protein- protein interaction. Identifying Protein-Protein Interaction In the post genomic era, few methods have proven especially helpful for this purpose. Yeast two hybrid (Y2H) system is one of them. In short, the Y2H method is designed to use a protein of interest as bait in order to discover proteins that physically interact with the bait protein, those proteins are termed as preys. In Y2H method, a single transcription factor is cut into two pieces called the DNA Binding Domain (DBD) and Activation Domain (AD), which stimulates the RNA polymerase to begin transcription.  Identifying Protein-Protein Interaction Fused to the DBD is the bait protein of interest (B), which cannot initiate transcription on its own. Fused to the AD the prey ORF, which can be any known or unknown protein. The prey protein of AD + ORF fused together cannot initiate transcription either. When the bait and prey proteins are produced in the same cell, they might interact; and if they do, transcription of His3 gene is initiated. Any ORF can be tested with Y2H, which means a proteome-wide survey can be performed rapidly by transforming a genomic library into cells that contain bait plasmids. In this way, every protein in a proteome can be tested individually for its potential interact with bait. Identifying Protein-Protein Interaction 15 Yeast two-hybrid assay Y2H  Yeast two hybrid assay Transcription factor, Gal4p, has DNA binding (BD)(aa1- 147) and transcriptional activator(AD)(aa768-881) domains Stimulates transcription at a promoter reconized by Gal4p (upstream activating sequence,UAS) Lac Z reporter gene encodes beta-galactosidase which produces blue pigment when the colony is grown in a media containing X-Gal Disadvantage: time consuming!  An example: Virion assembly The components come together and the Nucleocapsid is formed which in turn will become completed to the whole particle. The assembly process begins when concentration of structural proteins is enough within the cell to drive the process. Many protein-protein, protein-nucleic acid and in case of membrane viruses protein-membrane (fatty acid) interactions are needed. 18 The mechanisms of interaction Non-covalent so reversible Van del waals forces Hydrophobic interactions Electrostatic bonds Hydrogen bonds For strong couplings very accurate force field potentials are needed  How to study protein-protein interaction? Gel filtration FRET Fluorescence resonance Far western blot energy transfer microscopy Affinity chromatography Confocal microscopy Co-immunopercipitation 2 hybrid assay Co-crystallization for Protein microarry crystallography Maspec Capillary elecrophoresis NMR Nuclear Magnetic Biosensor Resonance Gel filtration chromatography Also called ”Size exclusion” Porous made up of cross- linked polymers Small molecules are trapped by the beads For self assembling proteins monomers come later Far western blot Also called ”Blot overlay” Fractionating proteins on SDS- PAGE Blotting to nitocellulose or PVDF membrane Overlaying with a solution of the protein of interest Binding the added protein to an immobilized protein on the membrane Detection with antibody against the overlaying protein  Co-Immunoprecipitation Protein A binds to antibodies Sepharose beads coated with protein A Specific antibody binds to the protein of interest The complex is precipitated by binding to the beads via protein A Proteins are released from beads by boiling Western blot Affinity chromatography In the case of His- tagged proteins The His-tagged protein binds to nickel or cobalt column His-tagged protein and it’s associated protein are eluted from the column by adding imidazole  Protein microarray A protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function. The proteins are arrayed onto a solid surface such as microscope slides, membranes, beads or microtitre plates.