Protein Interactions
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List some classes of DNA binding proteins
Leucine zipper motifs Zinc fingers Basic helix loop helix B-sheet
Where do most DNA binding domains interact with DNA?
With the major groove
Describe the basic properties of Leucine Zippers
Two alpha helices binding domains Form an inverted Y structure where each arm mediates binding to a specific DNA sequence in the major groove of DNA (TGTT) Each alpha helix bind to one hald of symetric DNA structure
Describe the basic properties of Zinc Fingers
Recognises DNA by using the 3 zinc fingers of cys-cys-his-his type repeats The three fingers have similar amino acid sequences The zinc finger holds together an alpha helix and beta sheet
Describe the basic properties of basic helix loop helix
Two monomers are held together in a four helix bundle: each monomer contributes two alpha helixes connected by a flexible loop of protein A specific DNA sequence is bound by two alpha helixes that project from the four helix bundle
Give three examples where metal is bound in proteins
Structural eg in zinc finger Regulatory eg in calmodulin Catalytic eg zinc, iron and copper
How is zinc coordinated in the zinc finger proteins?
Tetrahedrally Usually by cysteine or histidine residues - these proteins grab the zinc
At which position in the zinc finger protein can you find the cysteine and histeine residues?
Cys = 3 and 6 His = 19 and 23
Each zinc finger recognises how many nucleotides?
2
Which assay is used to study the strength and specificity of a proteins interaction with different DNA sequences?
Gel-mobility shift assay
What is the purpose of DNA footprinting?
Determines the DNA sequence recognised by a gene regulatory protein once it has been purified
How does DNA footprinting work?
It is based on nucleases or chemicals that randomly cleave DNA at every phosphodiester bond A bound regulatory protein blocks the phosphodiester bonds from attack, thereby revealing the proteins precise recognition site The footprint is where no cleavage occurs
What is the role of the EF hand?
Binds calcium or magnesium in structural signalling mdoe
What does the SH2 domain bind?
Phosphoryated proteins
What does the SH3 domain bind?
Proline rich motifs
What does the PH domain bind?
Phosphorylated lipids
What are the steps in the signalling pathway?
1) Activated receptor phosphorylates itself on tyrosine 2) It recruits insulin receptor substrate 1 via a PTB domain 3) The PH domain of IRSI binds to phosphoinositides on the plasma membrane 4) The activated rceptor phosphorylates IRSI on tyrosines 5) One phosphotyrosine binds the SH2 domain of GRB2 (adaptor protein) 6) GrB2 then uses two proleine rich SH3 domains to bind to two different proteins: - SOS - Scaffold protein
How can we study protein protein interactions biochemically?
Centrifugation Chromatography Pulldown reactions
How can we study protein protein interactions structurally?
X-ray crytallography NMR Electron Microscopy
How do you tag proteins of interest?
Tag the protein of interest eg use flag peptide (DYKDDDK) Then use antibody against the tag to track the protein
List two common tags for affinity chromatography/pull down
Glutathione -S-transferase (GST) Hexahistidine (6xHis)
List three common tags for immunoprecipitation
HA peptide MyC peptide Flag peptide
What are the steps involved in GST affinity pulldown?
1) Protein X bound to GST coated beads 2) When cell extract is added interacting protein bind to protein X 3) Glutathione solution elutes the fusion protein together with the interacting protein
What is the yeast two-hybrid screen?
Where bait and prey proteins are bound to DNA binding domains and transcriptional active domains When 2 proteins interact the reporter gene is switched on