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Protein-protein interactions Three types of interactions • Interactions between domains in multidomain proteins • Interactions between the domains in stable complexes • Interactions between proteins in transient interactions Stable complexes • The proteins in stable complexes tend to be more closely co-expressed and more closely conserved compared to proteins in transient interactions and monomeric proteins Yeast-two-hybrid screens • Uses the transcription of a reporter gene driven by the Gal4 transcription factor to monitor whether or not two proteins are interacting Yeast-two-hybrid screens Gal4-AD A B RNApol Gal4-DBD DBD = transcription factor AD = activation domain Yeast-two-hybrid screens Gal4-AD A B Reporter gene RNApol Gal4-DBD DBD = transcription factor AD = activation domain Computational methods • Conservation of gene order – In prokaryotes, genes are organized into operons of co-regulated and co-expressed groups of genes – Genes that are consistently part of the same operon across different, distantly related genomes are likely to be part of the same protein complex or functional process across all species – They have been selected to remain as a co-regulated unit throughout the extensive shuffling of gene order that takes place in prokaryote genomes Computational methods Computational methods • Gene fusions – Look for cases across a set of genomes where two or more orthologs are part of the same gene in one genome, presumably as a result of gene fusion – The prediction is that the orthologs that are separate genes in other genomes interact with each other – Fused proteins are not only co-regulated, as in the conservation of gene order, but also permanently colocalized in the cell – This method is limited to certain classes of proteinprotein interactions: • members of the same stable complex • proteins in the same metabolic pathway Computational methods yeast E. coli Computational methods • Phylogenetic methods – Relies on the detection of homologs in a set of genomes – If the pattern of homolog presence or absence is the same in a group of proteins, then these proteins are clustered together as belonging to the same functional class Computational methods Predicted to interact Gene 1 Genome 1 Genome 2 Genome 3 Genome 4 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 Protein chips • Miniature devices on which proteins, or specific capture agents that interact with proteins, are arrayed • Protein chips can act to – separate proteins on the basis of specific affinity – characterize proteins if the capture agent is highly specific (e.g., antibodies) • Advantage: ultra-high throughput analysis Protein chips • Antibody chips – Arrayed antibodies used to detect and quantify specific proteins • Antigen chips – Arrayed protein antigens used to detect and quantify antibodies • Universal protein arrays – Any kind of proteins arrayed to detect or characterize proteinprotein and protein-ligand interactions • Protein capture chips – Arrayed non-protein capture agents • Solution arrays – Coded microspheres or barcoded nanoparticles next generation Protein chips