Km Usuhs Class Improved1.2 Copy

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Protein-protein interactions
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Km Usuhs Class Improved1.2 Copy

  1. 1. INTERACTIVE PROTEOMICS – TECHNIQUES FOR EXPLORING THE SOCIAL NETWORK OF CELLS Karobi Moitra (Ph.D) NCI Frederick , NIH Cancer Inflammation Program Human Genetics Section Frederick MD.
  2. 2. Proteome: the entire protein complement of a cell , tissue, or organism The proteome is DYNAMIC !
  3. 3. Why is the proteome dynamic ? Proteins can be: Synthesized Modified by post-translational modifications Undergo translocations within the cell Degraded
  4. 4. Examination of the proteome of a cell is like taking a “snapshot” of the protein environment at any given time
  5. 5. FREEZE FRAME………
  6. 6. Proteome: the entire protein complement of a cell , tissue, or organism Proteomics: is the large scale characterization of this proteome
  7. 7. Why do we need to characterize the proteome? • To obtain a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually • To create a complete three-dimensional (3-D) map of the cell indicating where proteins are located
  8. 8. The Google Earth Analogy Global Protein Landscapes
  9. 9. Different areas of study are now grouped under the rubric of proteomics include: Protein modifications Protein function Protein localization Protein-protein interactions (Interactive proteomics)
  10. 10. WHAT IS INTERACTIVE PROTEOMICS OR PROTEIN – PROTEIN INTERACTIONS? THE ABILITY OF A PROTEIN TO BIND OR INTERACT WITH ANOTHER PROTEIN OR PROTEINS Types of protein interactions : Permanent interactions Transient interactions
  11. 11. From the need for more -omics came the term interactome
  12. 12. THE INTERACTOME COMPLETE PROTEIN INTERACTION NETWORK OF A CELL OR AN ORGANISM Just as humans don’t thrive when isolated from other humans - the same can be said for proteins !
  13. 13. WHY ???
  14. 14. Proteins interact with other proteins to provide : Structural integrity to the cell (e.g., actin filaments) Transport molecules (e.g.,Transporters) Propagate signals (e.g., kinases) Transcribe DNA, translate other proteins etc.
  15. 15. ….there is no protein discovered yet that acts on its own without interacting with any other entity !
  16. 16. The entire protein complement of a cell, tissue, or organism is called the PROTEOME The proteome is DYNAMIC Proteins can interact or bind with other protein(s) This ‘social’ network is called the INTERACTOME
  17. 17. ….in the real world you have to interact with people to learn the ‘social dynamic’ …. in the protein world you would have to know how proteins (and other components of a cell) interact with each other in order to explore the ‘cellular’ dynamic
  18. 18. You have been asked to find the interacting partners of a protein named ‘C3PO’. Your first task is to find out everything you need to know about this protein in order to undertake this study. Your tool is the internet, which sites you would go to and what information might you obtain from these sites to get the relevant background knowledge you would need to carry out the study?
  19. 19. Partial List of potential websites: www.google.com www.ncbi.nlm.nih.gov/ http://www.ensembl.org/index.html www.expasy.ch http://www.expasy.org/links.html
  20. 20. And a lot more links from this page………
  21. 21. 1.Clone and express the protein (C3PO) in an expression system of your choice 2. Optimize protein expression 3. Decide which techniques you would use to study protein-protein interactions
  22. 22. TECHNIQUES USED TO STUDY PROTEIN-PROTEIN INTERACTIONS
  23. 23. A. Standard techniques to probe protein-protein interactions Affinity purification Mass Spectrometry Two-Hybrid Assay Phage Display B. In Vivo Imaging Fluorescence Microscopy C. Biophysical Approaches Protein Co-crystallization D. Microarrays High Density Protein Microarray E. Computational/Bioinformatics Methods Computer programs that simulate protein-protein interactions Prediction of co-evolved protein pairs based on similar phylogenetic trees
  24. 24. A. Standard techniques to probe protein-protein interactions Affinity purification Basic Principle: Historically affinity purification was based on a specific biological interaction such as enzyme-substrate. In a broader sense it may mean Chemical/biological affinity. Stationary/solid phase Dynamic/liquid phase Immunoprecipitation Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.
  25. 25. Immunoprecipitation/coimmunoprecipitation Basic Principle: B lysate X A Y B A X Y B Cell lysis X A Freeze thaw Y Lysis buffer Hypotonic Mild detergent ProteinA/G beads (Ripa, NP40) X (binds to Fc of Ab) A Y Post- ip Run gel Visualize protein Excise band elute wash Digest Lysis buffer A X Low pH(change pH) MS SDS loading buffer Y
  26. 26. Disadvantage: An antibody to the specific protein of interest is required Solution: We can tag our protein of interest with an epitope tag
  27. 27. Epitope Tagging : Antibody recognizes a specific portion of the protein - epitope. Target protein Flag tag Anti-Flag Ab coupled to beads Associated proteins (i) Single Tag FLAG tag , c-Myc tag, GST tag, His tag etc. (ii) Tandem affinity purification TAP tag
  28. 28. (i) Single Tag
  29. 29. Attaching the Tag : Note: Tags can be N terminal or C terminal depending on where the functional region of the protein is located. Tagging close to the functional region may interfere with binding sites. Clone into vector Transfect into cells to express the protein
  30. 30. Histidine Tag Imidazole groups Imidazole can form a coordinate covalent bond with metals groups Nickel column Or Imidazole
  31. 31. Transfect cells Controls : 48-72hrs Transfection: (for peptide purification- Untransfected cells (Complex pulldown) antibody production Vector transfected Also complex pulldown) Known positive control Pulldown: Vector transfected cells Known positive control Or Mechanical lysis Freeze-thaw method His-tagged protein binds to Ni column (Low conc. to wash out non-specific binding) (Compete off His tagged Protein)
  32. 32. (ii) Tandem Affinity Purification : 2 step purification : 1. Purify through Protein A tag on a IgG- sephrose column 2. Purify through Calmodulin binding domain on a Calmodulin-sepharose column 2 step purification removes a lot of the background / non-specific protein binding
  33. 33. Tandem affinity purification (TAP) TAP TAG (tobacco etch virus)
  34. 34. IgG-sepharose bead Step 1 TEV cleavage Purify protein by passing through IgG column and elute with TEV IgG-sepharose bead Step 2 Purify protein by passing Through Ca + calmodulin column And elute with EDTA Elute (EGTA) IgG-sepharose Target protein bead Calmodulin-sepharose beads
  35. 35. The TAP strategy
  36. 36. Evaluation of a Co-IP Captured interaction 1.Confirm that the co-precipitated protein is obtained only by the antibody against the target , try and use monoclonal antibodies , if using polyclonals purify the antibody using an affinity column containing pure target 2. Use an antibody against the co-precipitated protein to co-IP the same complex 3. Determine that the interaction takes place in the cell and not as a consequence of cell lysis, use co-localizatiion or mutation studies to confirm interaction. 4. Run a negative control IP with unrelated antibodies.
  37. 37. (i) Single Tag  (ii) Tap tag  (iii)Photochemical/chemical crosslinking
  38. 38. Photochemical / Chemical Crosslinking of Proteins The interactions or proximity of proteins can be studied by the clever use of crosslinking agents. Protein A and B may be quite close to each other in a cell and a chemical crosslinker can be used to probe the protein-protein interaction by linking them together, disrupting the cell and detecting the crosslinked proteins. B A
  39. 39. Diazirine based photo crosslinking Cells grown with photoreactive diazirine compounds Diazirine incorporated into protein UV light A B Diazirines activated and bind to interacting proteins (within a few angstroms)
  40. 40. Chemical Crosslinking • Covalently links distinct chemical functional groups & can detect both stable and transient interactions • If 2 proteins physically interact with each other they can be covalently crosslinked Crude cellular extract + crosslinking agent (maleimides -SH reactive groups would form disulphide bonds between proteins ) IP A B Recover complexes Cleave with DTT, BME which would break disulphide bonds Example : SMCC, succinimidal trans -4 (maleimidemethyl) cyclohexane-1- carboxylate
  41. 41. (a) Epitope tagging (b) Chemical cross-linking (c) TAP tag approach.
  42. 42. You have your putative protein -complex of interest how would you identify the individual proteins that make up this complex ?
  43. 43. SCHEMATIC DIAGRAM OF PROTEIN IDENTIFICATION (can ID only 50-60 aa)
  44. 44. MASS SPECTROMETERY Protein structural information : peptide mass amino acid sequences Type and location of post-translational modifications
  45. 45. Experimental Design Immuno-precipitation Examine complexes by SDS-PAGE Mass Spectrometry
  46. 46. LC- TANDEM MS In-gel digestion with trypsin (K/R) Extract peptides MALDI-TOF/TOF
  47. 47. PROTEIN IDENTIFICATION BY PEPTIDE MAPPING (MALDI-TOF) MALDI-TOF Matrix assisted laser desorption ionisation- time of flight Soft ionisation technique suitable for fragile biomolecules like peptides
  48. 48. Basic Principle of Mass Spectrometry How it works : The amount of deflection for a sideways force depends on the Mass of the ball (acceleration constant) Acceleration - known Force - known Mass - can be calculated Force= mass x acceleration
  49. 49. Peptides + matrix (matrix protects peptides from the direct laser beam and help absorption of laser energy) Spotted onto a target plate Ionised by laser beam (charge needed for deflection by electric field) Ionised particles enter flight tube Charged peptides move to other side of tube according to mass Peptides hit the detector and time of flight (TOF) is recorded (to calculate speed) Opposite charge (speed) (known force)
  50. 50. The computer generates a mass spectrum, with each peak representing the mass to charge ratio (m/z) as a function of the % relative intensity (abundance) of the detected peptide The list of experimental peptide masses is compared against the theoretical tryptic digest of every protein in a protein database. When the experimental data matches the theoretical, the protein is identified. A probability based scoring system is used for the search, indicating the ‘hit’ is not a random event.
  51. 51. PROTEIN IDENTIFICATION BY TANDEM MASS SPECTOMETRY (MS/MS) If the protein cannot be identifed via the peptide mass profile (eg the protein may not be listed in any database) then Tandem (MS/MS) may be used to obtain an amino acid sequence. Q1- 1st mass analyser (quadrupole) isolates peptide ion of interest Q2- Collision chamber peptide ion collides with neutral gas molecules (helium,nitrogen or argon) and fragments into smaller pieces Q3- 2nd analyser (TOF) leads to detector which gives a product profile (aa sequence) Fragments the peptides into the smallest length to ID short sequences
  52. 52. A. Standard techniques to probe protein-protein interactions Affinity purification  Mass Spectrometry  Two-Hybrid Assay Phage Display B. In Vivo Imaging Fluorescence Microscopy C. Biophysical Approaches Protein Co-crystallization D. Microarrays High Density Protein Microarray E. Computational/Bioinformatics Methods Computer programs that simulate protein-protein interactions Prediction of co-evolved protein pairs based on similar phylogenetic trees
  53. 53. Yeast 2-hybrid assay • Test the association of two specific proteins that are believed to interact on the basis of other criteria. • Define domains or amino acids that are critical for the interactions of two proteins that are known to interact • Screen libraries for proteins that interact with a specific protein.
  54. 54. Activating Basic Principle of 2- Hybrid Assays domain Binding domain The basic premise of a 2- hybrid assay is that a prey protein is detected with the help of a bait protein. A transcription factor is split into 2 parts a DNA binding domain - BD and an activation domain AD. The BD is engineered to bind to the bait and the AD is engineered to bind to the prey. Only if the bait and the prey protein interact will the transcription factor come together and transcribe a reporter gene.
  55. 55. Probing Protein-Protein Interactions with the Yeast 2-Hybrid Assay DNA Binding Domain Protein X + Activation Domain Protein Y YES NO HIS3 HIS+ his- lacZ Blue white ADE2 Whit red
  56. 56. Split-Ubiquitin Membrane Yeast Two-Hybrid System Drawbacks of typical Y2H necessitated the split-ubiquitin Y2H 1.Hybrid proteins are directed towards the nucleus so proteins that fold incorrectly in nucleus are excluded from the method (integral membrane proteins). 2. Interactions dependent on post-translational modifications ( in ER) won’t take place. 3. Interactions mediated by the amino-terminus may not work because the transcription factor domain blocks accessibility.
  57. 57. Split-Ubiquitin Membrane Yeast Two-Hybrid System 1. Contains 2 fragments of ubiquitin brought together upon interaction of the 2 proteins. Prey Bait 2. The bait protein X is fused to the C-term of ubiquitin (Cub) followed by a TF 3. The prey protein Y is fused to N-term of Y X ubiquitin (NubG) 4. The 2 plasmids are introduced into yeast L40 strain. Transcription factor 5. Interaction of X and Y leads to the assembly of ubiquitin and the proteolytic release of transcription factor (by ubiquitin proteases). 6. The transcription factor activates the 2 reporter genes lacZ and His3 so the interactions can be monitored by growing yeast in histidine deficient media or by performing an X-gal test for the expression of beta galactosidase.
  58. 58. A. Standard techniques to probe protein-protein interactions Affinity purification  Mass Spectrometry  Two-Hybrid Assay  Phage Display B. In Vivo Imaging Fluorescence Microscopy C. Biophysical Approaches Protein Co-crystallization D. Microarrays High Density Protein Microarray E. Computational/Bioinformatics Methods Computer programs that simulate protein-protein interactions Prediction of co-evolved protein pairs based on similar phylogenetic trees
  59. 59. PHAGE DISPLAY Basic Principle: In phage display new genetic material is inserted into a phage gene and the bacteria process the new gene so that a protein/peptide is made and exposed on the phage surface (due to a tag which only expresses on the cell surface). A population of bacteriophages display hundreds/millions of protein - one protein per phage.This is called a phage display library.
  60. 60. This library can be exposed to an immobilized target protein and some members will bind to the target. The immobilized target is then washed to remove non/loose binding phages. The DNA of phages that bind can be sequenced to identify the gene/protein.
  61. 61. B. IN VIVO IMAGING Fluorescence Microscopy Basic Principle Fluorescent molecules are irradiated with high intensity light. When these molecules absorb a photon of light an electron is boosted up to a higher energy orbit creating an excited state When this electron returns to the ground state a photon of light may be emitted- this is called fluorescence. Fluorophores have distinct excitation and emission spectra.
  62. 62. How can we use fluorescence microscopy to study protein-protein interactions?
  63. 63. 1. FRET (Fluorescent Resonance Energy Transfer) 2. BRET (Bioluminescence Resonance Energy Transfer)
  64. 64. 1. FRET (Fluorescent Resonance Energy Transfer) Normally an excited photon returns to the ground state when a photon is emitted. FRET results in the excitation of a nearby acceptor fluorophore which will emit a photon when it goes back to the ground state. The occurrence of FRET thus results in decreased donor emission and increased acceptor emission. Distance is everything ! FRET is extremely sensitive to the distance among fluorophores For CFP and YFP the half maximum distance or Forster radius is 49-52 angstroms
  65. 65. Basic Principle of FRET 475 CFP YFP One probable interaction partner is tagged with CFP the other with YFP. If the 2 proteins interact emission will be observed at 530nm instead of 475nm
  66. 66. Problems of FRET 1. Tissues and cells may be damaged by excitation light 2. Some tissues like the retina and most plant tissues are photoresponsive 3. Photobleaching, autofluorescence or diect excitation of the acceptor fluorophore may occur.
  67. 67. 2. BRET (Bioluminescence Resonance Energy Transfer) In BRET the excitation light is replaced by bioluminescent light from Renilla luciferase (RLUC) The luciferase is activated by its substrate coelenterazine. Bioluminescent light
  68. 68. C. Biophysical Approach 1. Protein Co-crystallization
  69. 69. Protein Co-crystallization -grow crystal -collect diffraction data -calculate electron density -trace chain & generate structure
  70. 70. SNL1 & YPD1 co-crystals SNL1 and YPD1 are part of a phosphorelay signal transduction pathway in yeast. these protein can be co-crystalized by using a phosphate analog (BeF3) which bind covalently and activates respose regulator proteins. (Chooback . L 2003)
  71. 71. Co-Crystal structure of YPD1 and SLN1 YPD - Yellow SLN1- Cyan (Xu et. al 2003)
  72. 72. D. Microarrays High Density Protein Microarray Microspots of the captured molecules are immobilized in rows and columns on a solid support They are exposed to samples containing the corresponding binding molecules. Proteins interact
  73. 73. Readout systems based on fluorescence, chemiluminescence,mass spectometry, radioactivity etc. can be used to detect complex formation
  74. 74. E. Computational/Bioinformatics Methods 1.Computer programs that simulate protein-protein interactions ie Docking programs like Autodock. 2. Prediction of co-evolved protein pairs based on similar phylogenetic trees This method involves using a sequence search tool such as BLAST for finding homologues of a pair of proteins, then building multiple sequence alignments with alignment tools such as Clustal. From these multiple sequence alignments, phylogenetic distance matrices are calculated for each protein in the hypothesized interacting pair. If the matrices are sufficiently similar they are deemed likely to interact.
  75. 75. A. Standard techniques to probe protein-protein interactions Affinity purification  Mass Spectrometry  Two-Hybrid Assay  Phage Display  B. In Vivo Imaging Fluorescence Microscopy  C. Biophysical Approaches Protein Co-crystallization  D. Microarrays High Density Protein Microarray  E. Computational/Bioinformatics Methods Computer programs that simulate protein-protein interactions  Prediction of co-evolved protein pairs based on similar  phylogenetic trees
  76. 76. Humans do not thrive when isolated from others - the same can be said for proteins !
  77. 77. TO MARGUERITE by: Matthew Arnold (1822-1888) ‘Yes in the sea of life enisled, With echoing straits between us thrown. Dotting the shoreless watery wild, We mortal millions live alone. The islands feel the enclasping flow, And then their endless bounds they know…’
  78. 78. Think-Pair- Share Activity In light of what you have learnt about proteins today do you think that a protein can function on its own isolated from other proteins? For or Against
  79. 79. ‘O then a longing like despair Is to their farthest caverns sent! For surely once, they feel, we were Parts of a single continent. Now round us spreads the watery plain-- O might our marges meet again!’

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