Protein Characterization and Function PDF

HSS 3109 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of...

HSS 3109 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of Ottawa Pavillon Roger Guindon Room 2135 451 Smyth Road, Ottawa, Ontario K1N 8M5 Lab website : www.burellelab.com Phone (office) : 613-562-5800 ext 8130 Protein purification, characterization and function analysis Learning objectives 2 • Be familiar with assays to determine protein concentration • Learn how electrophoresis works and how to visualize proteins using SDS-PAGE, Native PAGE, IEF-PAGE, 2D-GE • Understand the main methods used to analyse • Relative molecular mass • Primary structure • Secondary structure • Tertiary structure • Protein interactions Determination of protein concentration Know how much protein you were able to isolate/ was present in biological sample • A routine requirement during protein purification Linear relationship between protein concentration and optical density • Central to many techniques as several biological parameters are normalized per unit of proteins • Several methods: Lowry, Bradford, Bicinchoninic Acid (BCA) • All assays rely on changes in the color of a compound upon binding to proteins the more proteins there are, the more coloured compound ill be present in assay, and the darker the colour will be • Color changes can be assessed using spectrophotometric plate readers and protein concentration of sample deduced by comparing to a standard curve total protein assay BCA assay reaction • darker the purple colour, the more protein In sample • three replicates of the same sample - do an average of each well for reliable reading Visualization of proteins using electrophoresis • Electrophoresis is the seperation of macromolecules from complex mixtures by application of an electric field • Arne Tiselius won the Nobel Prize in Chemistry in 1948 • Principle: Macromolecules in the mixture will migrate at different speeds, depending on the nature of the gel and the characteristics of the macromolecules (mass Migrating and/or charge). through electrical field • Importance: A major advance in the ability to resolve and characterize proteins • Electrophoresis can be performed using different matrices (paper, various gels…) for protein seperation • Polyacrylamide Gel Electrophoresis (PAGE) is the standard methods in biomedicine PolyAcrylamide Gel Electrophoresis (PAGE) • Gels are: • Formed using chemical crosslinking of Acrylamine, and Bisacrylamide, initiated by ammonium persulphate and the base TEMED solifies in 10-15 mins • Generally formed as vertical “slab” gels Are more resistance than agarose gels • Referred to by the percentage of acrylamide in the gels Polymerizes w/ molecules of the same type and form a gel/matrix Changing size of pores in sleeve • The % acrylamide is varied depending on the nature and size of the macromolecule being separated 5-8% • Lower percentage gels: Nucleic acids, isoelectric focusing (seperation accroding to charge) gel is less porous • Higher percentage gels (above 10%: ProteinCan separate very separation according to mass (SDS-PAGE) small proteins • Gradients can be used to improve separation injection wells Ex. At top of gel, only 5% acrylamide, and then progressively, you go to 8%, 10%, 15%, and the bottom would be highest concentration - as molecules go down gel, they are getting thru smaller pore size of gel PAGE allows to: • Resolve proteins in complex mixtures • Estimate their size • Determine some of their properties Molecular Biomethods Handbook, Second Edition, JN Walker & R Ripley Editors, Humana Press 2088 very big Can easily separate 30 from 22, etc. SDS-PAGE most common variant • SDS: Sodium Dodecyl Sulfate is a negatively charged (anionic) detergent. • SDS linearizes proteins, and binds to them in proportion to the # of amino-acids. solubilizes protein, linearizes it and destroys all the chemical/covalent links in the protein and bind to the positively charged amino acid on the protein linerized protein w/ uniformly negative charge • Thus all proteins become negatively charged • Mobility will only depend on size (inversely proportional to the log of molecular weight) Larger the size, smaller migration distance Smaller the size, the greater the migration distance allows us to see migration front - can stop electrophoresis when it reaches the bottom • Proteins can then be stained (Coomassie blue, Ponceau Red, Silver staining etc…) and their apparent molecular weight can be estimated based on the migration distance (Rf) vs that of mass standards On gel, you can add specific antibody to detect the presence of a specific protein in your sample and only measure it Theoretical travelling distance from top to bottom Covalent bonds Compare migration of protein of interest to molecular weight ladder SDS-PAGE Reducing and non-reducing SDS-PAGE • SDS-PAGE can be perfomed in reducing or non-reducing conditions Can do experiment with and without reducing agent to examine associations between proteins • Reducing conditions: agents that reduce disulfide bonds, (such as 2-mercaptoethanol) These help protein folding are added to reduce covalent bonds within a protein (improves linearization) and between multimeric protein complexes. Subunits assembly of Ig is maintained Together have molecular weight of around 150 kDa subunits that are attached together and form an intact protein • Use of reducing conditions allows a more accurate estimate of protein size • Comparing a sample under reducing vs nonreducing conditions allows to tell if a protein of interest might be covalently associated with another protein within a cell if under reducing and non-reducing conditions the migration pattern is the same, then you know the protein wasn’t associated with another one covalently in vivo Around 55-60 KD Around 25 KD no longer see 150KD molecule since disulphide bond linking light and heavy changes have been destroyed Allows us to conclude that immunoglobulins are composed of multimeric protein - light + heavy chain covalent bonds maintained Native PAGE • Proteins are maintained in their native state following extraction with mild detergents (DDodecyl-Maltoside or digitonin) Transmembrane protein Minimal amount of detergent to get rid of lipid bilayer and extra protein complex w/o disturbing structure Mild Extraction Denaturation (SDS) Study each one separately Lipid bilayer • Proteins can be separated in absence (clearnative) of presence of Coomassie blue (blue native) to separate on size and charge or size alone. Protein complex composed of 4 subunits Native protein complex folding and association between subunits is kept —> activity of complex is maintained Everything is blue • NATIVE-PAGE: • Preserves protein-protein interactions that normally exist in vivo in cells protein is not • Allows to recover proteins in functional since “totally” disrupted form (e.g. with retained enzyme activity) • Can be used as a first separation for 2-dimensional gel electrophoresis (see next slide) Abolishes differences in charges between proteins • Complexes are separated in the first dimension in native gels, and then in the second by reducing SDS-PAGE. Linearized dissociated proteins clear buffer Native (non-denaturing) PAGE gels 2 Dimentional-PAGE 2 successive rounds of electrophoresis • Dramatically improve separations and resolution of complex mixtures of of the best ways to assess how proteins. One proteins interact in vivo • Separation in the first dimention can be perform using Native-PAGE or IEF-PAGE Mild Extraction Denaturation (SDS) Lipid bilayer Native protein complex • Gel is flipped and run in 2d dimention under denaturating conditions (SDSPAGE) SD Add S Once you stop the migration, you cut each lane of your gel, flip it 90 degrees and put it on top of an SDS PAGE gel (instead of loading samples) • when you start electrophoresis, proteins from the native PAGE will start migrating in the SDS PAGE blot Load native protein complex on top of native PAGE gel SDS-PAGE NATIVE-PAGE or IEF-PAGE protein complexes migrate on the gel and they will remain with this structure Individual components of the protein complex being seperated • can identify how many proteins form a protein complex 2 Dimentional-PAGE Native (non-denaturing) PAGE gels Mitochondrial respiratory chain has 4 protein complexes and ATP synthase • in vivo, they form multi-protein complexes called super complexes IVn III2 Use antibodies against each complex to identify which units make up each super complex dimers of complex 3 w/ association of complex 4 III2-IV I-III2 Dimers of complex 3 I Supercomplexes formation I-III2-IVn II Respirasome III AT P sy n IV Most complex unit • mixture of 1,3,4; functionally and structurally attached together • if isolated form mitochondrial membrane, it can respire alone ATPsyn2 Monomeric OXPHOS complexes Each band coreresponds to a supercomplex I IV III V With disease, these molecular super complex dissociate and the respiratory chain becomes less effective, and you start having ATP depletion syndromes IsoElectric Focussing (IEF)-PAGE In vivo, proteins also have an electrical charge based on the composition of their amino acids; some amino acids have a positive charge, others have a negative charge • some chemical modifications of proteins alter their charge; e. Glycotilate a protein or add a phosphate to it, it will change its electrical charge since phosphate is negatively charged • • Separate proteins according to their isoelectric points (pi) basic - low proton concentration at the top The pi of a protein is defined as the pH at which it has no net charge Not commonly used Useful for analyzing protein modifications that cannot be picked-up by SDS-PAGE such as: very small changes in the weight • Glycosylation status Acidic at the bottom - high proton concentration Gel has pH gradient Adding a couple of sugars • Phosphorylated isoforms 1st dimension (IEF) one of the most common posttranslation modifications of a protein • huge regulatory factor • Can be used as a first separation for 2dimensional gel electrophoresis followed by SDS-PAGE. Instead of separating by mass, you can speerate initially by isoelectric point 2nd dimension (SDS) • Load proteins in the middle of gel at physiolo gical pH of 7 When you turn on current, proteins will migrate depending on their net charge • negative proteins migrate towards positive, and vice versa • stop when elec. charge is equal to that of the gel • can determine net charge depending on migration distance Identification of proteins by gel staining Staining: Autoradiography: Immunoblotting: Labelling with 35S-Methionine amino acid that contains sulfur • can label sulfur w/ radioactive isotope of sulfur so that it becomes easily visible on a netoreography Ponceau Sensitivity + Coomassie blue time Allows to monitor Allows to visualize dynamic changes specific proteins Most sensitive stain over time: Incubate w/ primary antibody against target protein, then secondary antibody w/ signal Protein synthesis, Silver staining complex and you would see a single band somewhere in the gel that corresponds to can resolve total protein and see degradation, stability, location of protein of interest entire andiron pattern of your gel transport Culture cells in culture media that contains S35-methionine Stains all proteins • as cells synthesize protein, they incorporate S35 in the proteins and become labelled resolve every protein in your gel • ranked by sensitivity • do experiment over 24h; each 5-6 hr, take a sample and run a gel; do an x-ray; we would see increasing amount of S35 signal, and rate of accumulation overtime would indicate rate of protein synthesis Characterization of protein structure Specific amino acid sequence composing the protein • linearized string of amino acids Chemical bonds form between amino acids Different types of 3D structures • first level of 3D organization Regions of protein fold together to make polypeptide chain w/ more complex structure Make multi subunit protein • this example is composed of 4 subunits of protein Characterization of protein structure More popular Characterization of protein structure The old school: Sanger method Sequential labeling and cleavage of N-terminal amino acid Have purified protein react w/ a label (DNFB) - labels last amino acid of polypeptide sequence label N-terminal AA Incubate in presence of H3O+ Labelled amino acid is released All peptide bonds between amino acids are broken up DNP amino acid is identified Migrate on a gel comparing it with a known standard DNP-amino acid using gel chromatrography Then restart, and label next amino acid, etc. Sir Frederick Sanger Nobel prize 1958 Labor intensive!!! Milligram amounts of starting material are required Required very large amounts of purified protein of interest for sequencing • Severly limited • ex. Purifying a hormone - need to “hack” many animals to get appropriate amount for sequencing Characterization of protein structure We can identify 500-1000 proteins in mixture of cellular homogenate Proteins are first separated by 1D or 2D PAGE Extract band Trypsin is commonly used. It always cleave proteins between Lys and Arg residues (i.e. predictible) makes it easier to “put things back together” Seperate by gel electrophoresis Lyse cells Matrix-Assisted Laser Desorption/Ionization (MALDI), and ElectroSpray Ionization (ESI) are typically used to ionize proteins fragments allows for detection in mass spectrometer Separate according to size small ones leave device first, large Gives electrical charge ones delayed two mass spectrometers, back to back Directly “injected” in mass spectrometer Disrupted into individual amino acids Peptide mass fingerprinting Looks at relative abundance of MS spectrum • each peptide has a “signature” High throughput Nano-pico mole amounts are required De novo sequencing if the protein is not in the database, tandem MS/MS is used to sequence peptide fragments peptide masses are compared with a protein database if it matches, protein is identified Characterization of protein structure Shoot electrons on cyrtslaiized molecule • electrons are defracted and will hit the film • map refraction pattern or secondary structure (if it has alpha helices, or beta sheets, etc.) • To solve the tertiary structure of proteins two traditional approaches have been used: • X-Ray crystallography • • purify protein of interest w/ highest purity possible in native state then crystallize molecule to form solid very complicated as some proteins don’t crystallize • Nuclear Magnetic Resonance (for proteins that cannot be crystalized) Characterization of protein structure • High resolution cryo-electron microscopy is now able to resolve protein structure. • Sample is bombarded with electron in a vacuum To visualize them • Scattered electrons are focused on the microscope lens and imaged. • Sample is tilted at various angles relative to electron beam and a series of images is taken can do this to resolve proteins if electron microscope has appropriate magnification • Images are used for 3D-redonctruction Pseudo-3D image Characterization of protein structure Very similar to human one Mitochondrial tomography of ATP synthase can cut open volume and resolve single slices and look at internal structure of the protein Characterization of protein interactions Protein-Protein Interactions Several approaches can be used to study proteinprotein interactions • • • • • • • • • Yeast two-hybrid screen Non-reducing/non-denaturing gels and gradients Co-immunoprecipitation Far western blot Pull-down assays Chemical cross-linking BioID Fluorescence resonance energy transfer (FRET) Chromatin immunoprecipitation (CHIP seq) Characterization of protein interactions Co-immunoprecipitation some proteins associated together • Broadly used • An antibody (monoclonal or polyclonal) And potential proteins that interact w/ protein of interest against a specific target protein forms an immune complex with that target in a sample • The immune complex is precipitated on a beaded support to which an antibodybinding protein is immobilized (such as Protein A or G), • The antigen is eluted from the support and analyzed by SDS-PAGE • Can detect interaction that are direct or indirect since Co-IP will frequently precipitate protein complexes. Add antibodies coupled to agarose or magnetic beads • antibody specific to purple protein • spin to pull down purple protein and proteins that interact w/ it Identify by electrophersis and western blot Characterization of protein interactions Co-immunoprecipitation Artifacts Real interaction Target protein Co-IPed w/ protein that is really associated w/ it • Input: sample used for IP Entire sample before IP • Unbound: what is left after removing the beads • IP: the beads + bound proteins • Y Y real interactions Y • Several controls need to be performed to make sure that a given interaction occurs between the protein of interest and the coimmunoprecipitated protein. need to be able to operate artifacts from Two unspecific proteins stuck to bead and antibody dosen’t detect anything Non-specific binding Random protein got stuck on the bead and got pulled down Control Ab is used to test this Everything remaining at surface is unbound fraction that you pulled down, rinsed, and eluted wouldn’t expect to see myc or co-immunoprecipitated protein if it is a real interaction myc highly enriched in IP compared to input • proof that you were able to immunocapture This is a successful IP sinc3 co-immunoprecipitated protein isn’t attached to a bead randomly and is only coming down w/ myc Typically run all three fractions together in western blot • this western blot uses antibody against myc Presumably associated w/ myc and pulled down by immunocapture Characterization of protein interactions BioID method • Tracks the interaction partners a protein has had within a cell (a history of its interacting types of interactions - protein can bind to one protein, then partners). many dissociate and bind to another, etc. Proteomics • Fundamental difference with co-IP is that the method can detect transient interactions need to be interacting at the moment you lyse the cell, between proteins Don’t and you can still know that there was an interaction • Based on proximity-dependent biotinylation of proteins by a promiscuous biotin ligase mutant BirA (R118G), which is fused to your protein of interest. • After a period of incubation, biotynylated proteins are purified and analyzed. Transfect cells with a viral vector or plasmid encoding protein of interest coupled to enzyme BirA • when protein comes close to binding partner, BirA will take biotin and add biotin to the protein thereby labelling it • at the end of fixed timepoints, you lyse the cells and purify all biotin labelled proteins using streptavidin beads • can tell that these proteins interacted w/ protein of interest at certain times in the cell culture Characterization of protein interactions Fluorescence Resonance Energy Transfer (FRET) • FRET relies on the distancedependent transfer of energy from a donor molecule to an acceptor molecule and the resulting emission of fluorescence by the latter. • always involves two fluorophors • Donor and acceptor fluophores are attached to proteins for which we wish to examine interactions No fluroscne emitted if two flurophors are distanced efficiently Fluorescent light emitted by one flurophor will excite the other, causing it to emit fluorescent light too Can be used to detect change in conformation of proteins (ex. Conformation in G proteins (activation)) • This method therefore relies on a priori knowledge of potential interactions between candidate proteins can measure distance between two interacting proteins based on fluroscnece intensity Labelling two proteins that you already expect to interact • not a good tool to discover new interactions Tag two different proteins, if they interact together, you will get a FRET signal Characterization of protein interactions Calcium binds to calmodulin when it comes into the cell Fluorescence Resonance Energy Transfer (FRET) Activates kinase thru phosphorilation Detect calcium wave contractions in smooth muscle cells Interactions begin Interactions spread and amplify MLCK enzyme adds phosphate to myosin when active prevents myosin interactions Phosphorylation of regulatory light chain activates myosin head allowing for muscle contraction —> requires calcium Myosin head inactive • EGFP labeled calmodulin (Cam) • EYFP labeled myosin light chain kinase (MLCK) To trigger contraction Time(sec) pseudocoloured images - whenever there is green - no interaction between calmodulin and MLCK • when cell turns red, FRET signal, meaning calmodulin is bound to MLCK • contraction starts at right hand side of cell then after a couple of seconds, spread out and become more intensive • eventually whole cell will contract and calcium wave will start depolzariing connecting cell • Cell are exposed to stimuli that increase intracellular calcium waves (here going from top to bottom of cells). • Color change from green to red indicate time and space resolved protein-protein interaction between Cam and MLCK in live cells over time