Lecture 29 - Protein Interactions

Question 1

What are the 2 large categories of protein-protein interaction methods?

Answer 1

1. in the organism: immunoprecipitations -> separate proteins using antibodies, biotinylation -> tag proteins with biotin, FRET & BIFC -> fluorescence based approaches
2. in an unrelated organism system or in a test tube: GST-Pulldown -> test binding in a test tube, Two-hybrid -> bacteria or yeast

Question 2

How are proteins detected on a sample?

Answer 2

with electrophoresis and western blot
1. separate proteins by size
2. use a dye that reveals all proteins
3. use antibodies to detect specific proteins

Question 3

Co-immunoprecipitation experiments

Answer 3

used to detect protein-protein interactions
- antibody recognizes bait protein and causes complex of proteins to sink to bottom
- remove unbound stuff (i.e., protein still at the top)
- beads on bottom contain bait protein with interacting proteins
- reveal the proteins using western blot

Question 4

Why is western blotting not great?

Answer 4

• it’s time-consuming because you can only tag one target at a time with antibodies

Question 5

LC-MS/MS

Answer 5

• liquid chromatography - tandem mass spectrometry
• LC: separate components
• MS/MS: measures the mass-to-charge ratio (m/z)
• guess the identity from library of peptides
• allows identification of all proteins in the mix

Question 6

What should you do if there is no antibody available?

Answer 6

• fuse the protein to a tag (series of amino acids added to C terminus)
• antibodies for tags are always available
• His and FLAG are examples of tags

Question 7

Proximity biotinylation

Answer 7

• a new method that uses biotin ligase to mark proteins that are close
• fuse bait protein to a promiscuous biotin ligase (BirA)
• BirA adds biotin to exposed lysines on neighbouring proteins
• addition of biotin = biotinylation

Question 8

Streptavidin

Answer 8

• a proteinn produced by streptomyces avidinii with strong biotin-binding ability
• use streptavidin beads to separate biotinylated proteins

Question 9

Streptavidin column

Answer 9

• a way to separate biotinylated proteins
• biotinylated proteins will remain at column and rest will flow out

Question 10

What are 2 techniques we can use to determine where in the cell these interactions are happening?

Answer 10

1. FRET
2. Split-GFP/ BIFC

Question 11

Split-GFP/ BIFC

Answer 11

• fuse protein X to 1/2 GFP
• fuse protein Y to other 1/2
• if proteins are in close proximity/ interacting, the GFP halves will reassemble and fluoresce

Question 12

FRET

Answer 12

• fluorescence resonance energy transfer
• emission wavelength of 1 protein is excitation wavelength of the other
• if proteins are close enough, you will measure the emission of the second protein

Question 13

Disadvantages and advantages of FRET and BIFC

Answer 13

disadvantages:
- only 2 proteins at a time
- time-consuming
- need a fluorescence microscope

advantages:
- single cell resolution or better
- temporal resolution

Question 14

Explain the 2 hybrid systems in yeast

Answer 14

• GAI4 protein is cut into 2 parts: AD and BD
• one is fused to prey protein (GAI4 AD)
• other is fused to bait protein (GAI4 BD)
• if together, a functional GAI4 protein forms and activates transcription of the reporter gene
• GAL4 is a transcription factor and binds to UAS sequence

Question 15

A yeast two-hyrid (Y2H) experiment detects what?

Answer 15

• physical interactions of proteins through the downstream activation of a reporter gene
• i.e., if protein from the gene is transcribed, the proteins interacted (caused transcription of gene => translation of protein)

Question 16

Reporter gene makes yeast ____?

Answer 16

blue; pick the blue coloured yeast, sequence blue colonies to reveal the interacting proteins