Various assays

• Why is it important to identify protein-ligand interactions?

  • To see if they interact in the first place (yes or no)
  • To identify the specifics of the interactions for optimisation of drug development
• Ideal parameters for protein-ligand interaction techniques:

  • Quantitative - i.e. tells you about the binding affinity/saturation
  • Inert labels to ensure the label doesn't interfere with the native structure of the protein and to allow detection to be interference free
  • Ensure the sample is being measured in solution rather than bound to a solid phase as this maximises the amount of ligand that can bind to the protein
  • Sensitive and minimal sample size
  • High-throughput
  • Fast and atomic resolution
• How yeast 3-hybrid works

  • Similar principle to yeast 2-hybrid except it's used for identifying protein-RNA or RNA-RNA interactions.
  • Gene encoding POI is fused with LexA gene to make construct while RNA of interest is engineered to a section of RNA that binds to the MS2 binding site
  • Transform yeast
  • Interaction between the POI and the RNA causes MS2 to bind to the MS2 binding site, bringing the LexA DNA binding domain to the gene, allowing transcription
  • Again, use a selector gene such as lacZ
• What species of yeast is used for the Y2H and Y3H systems?

  Saccharomyces cerevisiae
• Pros of using the Y2H system for identifying PL interactions:

  • Scalable - can screen bait vs. many prey per experiment
  • Can screen cDNA library vs. cDNA library to figure out interaction maps between organisms (illustrates interactions between proteins, genes, metabolites etc. in a biological system)
  • Can directly identify the ID of interacting prey using the DNA sequence
• How glutathione S-transferase pull down works:

  • Construct of POI and GST is made and expressed
  • Bind fusion protein to glutathione coated beads on column
  • Elute column with prey protein - use a mixture
  • Wash off impurities using centrifugation
  • Elute fusion protein + bound ligands off the column using excess glutathione
  • Analyse using SDS-PAGE - the bound proteins should not migrate far due to the mixture containing more protein
• Pros of GST pull down assay:

  • Quick and easy if you have a clone of your bait available
  • GST-fusion bait protein is invisible on autoradiogram if radioactively labelled
• The gel shift assay identifies protein-nucleic acid interactions i.e. protein-DNA or protein-RNA (can be ss or ds)
• Methods by which DNA / RNA can be radiolabelled for the gel shift assay?

  • Incorporate [γ-32P]dNTP during 3' fill in reaction using DNA pol I (Klenow fragment)
  • 5' label with [γ-32P]dNTP using T4 polynucleotide kinase
• Native gel is used for gel shift assay as SDS-PAGE denatures proteins, so using that would mean the protein would no longer be associated with the DNA/RNA
• Gel shift assay gives information on stoichiometry as gives binding site information - according to distance migrated, you can tell if there are one, two or more DNA/RNA binding sites
• Pros of gel shift assay:

  • Easy to visualise result
  • Information on stoichiometry
• Biotinylation is the addition of a biotin molecule to your protein or ligand to identify protein-ligand interactions
• Benefits of using biotin specifically as a tag:

  • Covalently modifies biomolecules in specific, desired sites
  • Can modify proteins, peptide, RNA and DNA
  • Has minimal to no effect on protein structure so does not affect the protein-ligand interaction
  • Works in gentle conditions i.e. aqueous, near neutral pH
  • Widely commercially available
• How biotinylation takes place:

  • Gene construct is made of protein + Avitag (recognition sequence that acts as substrate of BirA (biotin ligase)
  • Expression of construct
  • Add biotin and BirA
  • Ligation requires ATP -> releases AMP + PPi
• Biotinylation of the protein of interest helps us to identify protein-ligand interactions because:

  • Biotin and avidin have the highest affinity of any protein and ligand - KD = 10-15 M, ΔG binding = -86 kJ/mol
  • Therefore if ligand is expressed with avidin in a fusion construct, binding of protein and ligand should cause biotin and avidin to bind too
  • Identify using ISC or fluorescent tag
• Some modified versions of avidin that make identifying P-L interactions easier:

  • NeutrAvidin - processed to remove carbohydrates so binding is more specific
  • Streptavidin - non-glycosylated so binding is more specific, less likely to induce unwanted immunological effects
  • CaptAvidin - has reduced binding affinity for biotin than regular avidin, making it easier to dissociate the avidin-biotin complex
• The biotin-avidin interaction is frequently exploited to identify PL interactions as the complex is stable to extremes of:

  • pH
  • Temperature
  • Organic solvents
  • Detergents
  • Denaturants
  • Even resistant to SDS-PAGE
• Desthiobiotin is an analogue of biotin that binds less tightly to biotin-binding proteins

  • Easily displaced by biotin
  • Therefore used as a way to add biotin to proteins without the need for enzymatic activity - just add biotin and it'll compete
• Cons of gel shift assay:

  • Takes hours
  • Not an equilibrium method i.e. KD cannot be measured
  • Doesn't work well for weaker interactions
  • Only semi-quantitative
• How the gel shift assay works:

  • DNA or RNA of interest is radiolabelled
  • Mix protein and radiolabelled DNA/RNA and run on native gel (i.e. polyacrylamide or agarose)
  • Negatively charged nucleic acid drives migration
• Cons of GST pull down assay:

  • Not very quantitative
  • Careful controls needed
  • May be susceptible to proteolysis (due to protease contamination)
  • Larger amounts of protein needed for identification
• Cons of using the Y2H system for identifying protein-ligand interactions:

  • Prone to false positives (due to variation in expression levels and non-specific interactions) so need to do more assays to confirm results
  • No quantification of affinity of interaction - just get a yes/no
• How yeast-2-hybrid system works

  1. Create two fusion constructs

  2. Yeast transformed with bait and allowed to proliferate before prey is introduced

  3. Use a reporter gene in one recombinant construct to confirm protein-ligand interaction
• Fusion construct 1

  Contains genes for the DNA binding domain of a gene activator protein (e.g. GAL4) and the protein of interest (bait)
• Fusion construct 2

  Contains the genes for the activation domain of a gene activator protein and the ligand (prey)
• If the protein of interest (bait) and ligand (prey) interact

  The activation domain and DNA binding domain will also interact to reform the gene activation regulator, resulting in expression of the reporter gene