Lecture 27: Functional Genomics 3; Protein-Protein Interactions

Question 1

What are proteins involved in?

Answer 1

1. Genetic pathways
2. Scaffolding
3. Enzymatic reactions
4. Multi-subunit machines e.g., RNA polym2

Question 2

What are the different type of protein-protein interaction?

Answer 2

1. Metabolic (enzymatic) pathways
2. Signalling pathways
3. Morphogenic pathways, in-which
   groups of proteins participate in the
   same cellular function during a
   developmental process
4. Structural complexes and molecular
   machines in which numerous
   macromolecules are brought together

Question 3

What is the importance of domains?

Answer 3

1. types of domains are important for 
   function
2. E.g., a kinase domain

Question 4

What is meant by “proteomics”

Answer 4

“large-scale study of proteins from a cell,

tissue or organism”

Question 5

Why study protein-protein interactions?

What are the two steps in understanding protein-protein interactions?

Answer 5

1. To understand the mechanism by which a
   set of proteins act together towards
   bringing about a common cellular
   function
2. Allows broader outlook on protein
   function and how biological processes
   occur
3. Two steps:
   a. identifying proteins that interact
   b. Determining the significance of the
   interaction network

Question 6

What are the different methods of studying protein-protein interactions

Answer 6

1. Yeast-two-hybrid (Y2H)
2. Biomolecular fluorescence
   complementation (BiFC)
3. Recombinant fusion proteins (protein
   tagging)
4. Pull down assays
5. Co-immunoprecipitation

Question 7

what is the “Yeast-two-hybrid (Y2H)” of studying protein-protein interactions?

Answer 7

1. Proteins expressed in yeast
2. Transcription factors have two domains:
   a. Binding domain (BD)
   b. Activation domain (AD)
3. In Y2H assay,
   a. wild-type GAL4: reporter gene
   expressed (AD & BD fused)
   b. Split GAL4: reporter gene silenced (AD
   & BD separated)
   c. Reconstituted GAL4: reporter gene
   expressed (proteins refused)

Question 8

What is Y2H bait and prey screening?

Answer 8

1. Bait protein (protein of interest)
2. Bait is expressed in fusion with the DNA-
   binding domain from the GAL4
   transcriptional activator
3. To provide the second hybrid protein, a
   cDNA-library is used that expresses
   cDNAs in fusion with the transcriptional
   AD of GAL4
4. Screening can then be done based on
   reporter gene expression

Bait = target protein
Prey = what proteins does bait interact with

Question 9

What are the pros and cons of Yeast-two-hybrid (Y2H)?

Answer 9

\+ easy
\+ fast
\+ no protein purification
\+ in vivo conditions
\+ can be adapted for high throughput 
   screens
\+ can detect transient interactions

• prone to false negatives
• prone to false positives
• not easy to be quantitative
• no control over post-translational
  modifications
  -only test binary interactions (although can
  do Y3H now!)

Question 10

What is bimolecular fluorescence complementation (BiFC)?

Answer 10

1. in vivo
2. Take fluorescent protein (e.g., YFP)
3. Chop it in half
4. Fuse to each half one of the proteins
   they think might interact (E.g., Y to
   YFP1 and X to YFP2)
5. If X and Y interact, yellow will fluoresce

Question 11

What are the pros and cons of bimolecular fluorescence complementation (BiFC)?

Answer 11

\+ in vivo assessment
\+ low cost and quiet simple
\+ no protein purification req.
\+ can simultaneously localise proteins in 
   cell

• need to be able to genetically manipulate
  your species of interest
• not easy to be quantitative
• only test binary interactions

Question 12

What are recombinant fusion proteins and why use them?

Answer 12

1. Fuse protein of interest “protein X” to
   fusion partner (e.g., GST, etc)
2. Fuse fusion partner to affinity ligand via
   affinity (e.g., GSH, etc)
3. This is allows use in an assay such as
   “Pull down assay”
   a. protein attached to GST
   b. column has GSH on walls to attach as
   runs through
   c. then elute with GSH, removing X-Y-
   GST from tube

Question 13

What is co-immunoprecipitation?

Answer 13

1. “uses an Ab to capture and purify your
   protein of interest, plus any interacting
   proteins”
2. Process
   a. lyase cells
   b. incubation with Ab that recognise
   protein of interest
   c. Wash to remove unbound protein
   d. Elute bound proteins
   e. Analyse further (e.g., mass spec to
   identify interacting proteins)

Question 14

What are the pros and cons of recombinant fusion proteins; pull downs & co-immunoprecipitations

Answer 14

+ powerful technique to demonstrate protein-protein interactions
+ Purified proteins can be used to assess biological activities in vitro

- Can be technically challenging to purify 
   proteins of interest 
- difficult to detect transient or weak 
  protein-protein interactions
- only test binary interactions
- Co0immunoprecipitation requires Ab