chapter6 protein interaction.pdf

· always complex interact Learning Outcomes In ~ & O Students should be able t...

· always complex interact Learning Outcomes In ~ & O Students should be able to : protein Understand the need for protein interaction studies in proteomics CHAPTER 6: understand the fundamentals of protein interaction tools Protein Interaction Studies understand the different information obtained from each analytical tool internet Toxin A experiment I ↑ the With eatmor Physical ee ni pote ~ immobile 1. Protein arrays (bio-chips, protein chips) Protein interactions Equivalent to DNA microarrays to detect ⚫ Proteins seldom act alone – usually work in a complex ⚫ a series of substrate (proteins, peptides, antibodies, other protein consisting of many proteins aptamers) – antibodies are the most common ⚫ Interaction with different proteins different functions? ⚫ spotted/ immobilised on an array know the protin interactions ⚫ sample added how toAnalytical Methods ⚫ binding of proteins in the sample to substrate on array is Q ⚫ Protein arrays detected glass side G ⚫ Sandwich immunoassay Initro celle lose O ⚫ Yeast two hybrid assay Can be used for ⚫ Immunoprecipitation ⚫ Screening/ profiling ⚫ Pull down assays ⚫ Test for protein-protein interactions ⚫ Fluorescence resonance energy transfer (FRET) ⚫ Test for nucleic acid-protein interactions ⚫ Test for protein-drug interactions ⚫ Test for enzyme-substrate interactions  positive Protein antibodies (protein) interaction ↑ G become Spot > immosaized - based on any ligand-binding assay that relies on the formation of product with an immobilized capture molecule and a target molecule present in the solution miniaturized and placed on a protein microarray chip common labels- Cytochrome Cy3 G O and Cy5, biotin, HRP I binding label Protein saml &If [ sys , cy5- Visualize interacted CMS Protein < pechrouchy - cannot > - cannot used antibody not validated Can be used to compare protein profiles of different tissue Many variations can be made : transcription samples e.g. cancer vs normal tissues by labelling with different ⚫ Ab chip to detect protein factor colors bioelente - ⚫ Ab chip to detect Ag ⚫ protein chip to detect interaction ⚫ DNA chip to detect protein that binds DNA e.g. transcription factors ⚫ Ligand / receptor chip to detect signalling proteins ⚫ Enzyme chip to test substrate Pictures from www.microarraystation.com immo  General protein anliyody ofhere term - 2. Sandwich immunoassay- e.g Antibody arrays used ⚫ e.g. an oligosaccharide chip can be used to capture proteins that binds to carbohydrate capture f visibl withou ↳ huq arsimes O cannot ye -sensitivit - used af & yo S e S a ⚫ Ab with different specificities immobilised onto a surface Technology is still new ⚫ Specificity of substrate is difficult to verify- not as clear as ⚫ binds specific proteins in sample protein-protein since oligosaccharide structure is complex ⚫ detect using second Ab ⚫ Background variations ⚫ many different proteins can be assayed on the same chip with toxin - able to bird molea Il el - d weal & 3. The yeast two-hybrid assay -donecate ⚫ Interaction between the ‘bait’ and ‘prey’ will bring the DBD and AD of GAL4 together Location a cur -uncens ⚫ Use to test if one protein an Al-activation / (‘bait’) interacts with other domain transpor proteins (‘prey’) ⚫ The bait protein is tagged to the DNA binding domain (DBD) of the yeast GAL4 transcription factor ⚫ A ‘library’ of prey proteins are tagged to the activation G domain of the GAL4 ⚫ DBD binds to the promoter > - GALY transcription factor senthesi metic se region of a reporter gene in a to 24 14 se Fag A yeast cell ⚫ Interaction reconstitute a functional GAL4 transcription interaction 3 of the reporter gene A will. occur protein of the reporter. gene me gene expression no interaction - no expression ⚫ Reporter genes, usually a few are used to reduce false ⚫ Gene for the ‘bait’ protein is cloned into a vector with a orie cor +ve DBD tag e.g. β-galactosidase ⚫ A cDNA library of ‘prey’ protein genes are cloned into a yeast colony is blue color when grown on X-gal second vector with an AD (activation domain) tag (A ⚫ antibiotic resistance gene cDNA library theoretically contains all the expressed genes of a cell) growth on medium with antibiotic ⚫ By co-transforming yeast cells with the ‘bait’ vector and ⚫ biosynthesis gene e.g. HIS3, URA3 one of the ‘prey’ vector – the interaction pattern of the growth on medium without histidine or uracil ‘bait’ protein can be studied PREY interaction study-protein-protein ~ hi surable 4. Immunoprecipitation c Using yeast two hybrid to investigate a protein interaction A-1.3 me microcentrifuge network ⚫ Capturing a multiprotein complex His know biological coxidation) B 1 - 3mL falcon by using an antibody to one of the ~ pathway tube ineviticton proteins in the complex bes co different I spirh ⚫ The other members of the complex can then be isolated and motiv Opeptide separate - - different identified using MS - wes intensity ⚫ Or analysed using western blot = sype ⚫ Results are then confirmed by doing a second precipitation using antibody to another protein in the M complex I Protein , capture another protein can capture  breakdown large protein > - trypsin of to small protein enzyme used - AD 5. Pull down assay ⚫ Similar to immunoprecipitation, but a bait protein is used instead of an Ab. ⚫ Other proteins that interact with the ‘bait’ will be captured ⚫ The bait can be tagged with a ligand and immobilised on a solid substrate ⚫ Captured proteins are eluted and MS a David attract G ab-only Hi - / Ms - after 2D SDS PAGE Me wo - ( campe tandem high resolution ms (double ms] protein separation - 6. Fluorescence resonance energy transfer (FRET) ⚫ Different types of strategy to capture a multiprotein complex non-radiative transfer of energy from an excited fluorophore (donor) to another fluorophore (acceptor) only occurs when molecules are in very close proximity (1-10nm) a valuable technique for studying interactions between molecules exciting the donor and then monitoring the relative donor and acceptor emissions, sequentially or simultaneously indicates when FRET has occurred can be used to determine when and where two or more molecules, often proteins, interact useful tool to quantify molecular dynamics in biophysics and biochemistry, e.g protein-protein interactions, protein-DNA interactions, and protein conformational changes GST- glutathione-S-transferase  common combination of fluorophores: cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), a donor- acceptor pair is used for studying protein-protein interactions. CFP is excited, two molecules within 10 nm of each other - energy may be transferred from excited CFP YFP causing the YFP to emit yellow light, detected independently of the blue light that excited CFP nor a most signe not Thank you