Protein Interaction Studies

Protein Interaction Studies

• Proteins seldom act alone, usually working in a complex consisting of many proteins.
• Interaction with different proteins leads to different functions.

Analytical Methods

• Protein Arrays (Bio-chips, Protein Chips)
  • Equivalent to DNA microarrays
  • A series of substrates (proteins, peptides, antibodies, aptamers) are spotted/immobilized on an array
  • Sample is added, and binding of proteins in the sample to substrates on the array is detected
• Sandwich Immunoassay
  • Capture antibodies with different specificities are immobilized onto a surface
  • Binds specific proteins in the sample
  • Detected using a second antibody
  • Many different proteins can be assayed on the same chip
• Yeast Two-Hybrid Assay
  • Used to test if one protein (‘bait’) interacts with other proteins (‘prey’)
  • The bait protein is tagged to the DNA-binding domain (DBD) of the yeast GAL4 transcription factor
  • A ‘library’ of prey proteins are tagged to the activation domain (AD) of the GAL4 transcription factor
  • Interaction between the ‘bait’ and ‘prey’ will bring the DBD and AD of GAL4 together, reconstituting a functional GAL4 transcription factor, leading to the expression of a reporter gene
• Immunoprecipitation
  • Used to study protein-protein interactions
• Pull-Down Assays
  • Used to study protein-protein interactions
• Fluorescence Resonance Energy Transfer (FRET)
  • Used to study protein-protein interactions, nucleic acid-protein interactions, protein-drug interactions, and enzyme-substrate interactions### Immunoprecipitation
• Immunoprecipitation involves capturing a multiprotein complex by using an antibody to one of the proteins in the complex.
• The other members of the complex can then be isolated and identified using Mass Spectrometry (MS) or analyzed using Western Blot.
• Results are then confirmed by doing a second precipitation using an antibody to another protein in the complex.

Pull-Down Assay

• The pull-down assay is similar to immunoprecipitation, but a bait protein is used instead of an antibody.
• The bait protein is tagged with a ligand and immobilized on a solid substrate.
• Other proteins that interact with the bait are captured, and the captured proteins are eluted and analyzed using MS.

Protein Identification

• Proteins can be broken down into smaller peptides using an enzyme like trypsin.
• The peptides can then be analyzed using MS, which can identify the proteins present in the complex.

Fluorescence Resonance Energy Transfer (FRET)

• FRET is a technique used to study protein-protein interactions.
• It involves the non-radiative transfer of energy from an excited fluorophore (donor) to another fluorophore (acceptor).
• This energy transfer only occurs when molecules are in very close proximity (1-10nm).
• FRET can be used to determine when and where two or more molecules, often proteins, interact.

FRET Applications

• FRET can be used to study protein-protein interactions, protein-DNA interactions, and protein conformational changes.
• A common combination of fluorophores used for studying protein-protein interactions is Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP).