Question

Topic: Protein interaction and phase separation (Subject: Functional Genomics and Bioinformatics)

Please answer both Part A and B if possible, IF NOT PRIORITISE ON PART B

Consider the following scenario. A mutation in gene M was discovered to be a highly penetrant risk factor for disease P. There is no information on what the function of gene M is or what other proteins the gene product of M interacts with. All that is known is that M is abundantly and ubiquitously expressed in all cell types.

A. Describe in detail one (1) experimental approach that can be used for defining what proteins bind to gene product M and how the mutation alters the binding partners.

B. If purified gene product M were found to phase separate into droplets in a test tube, describe an experiment that that would distinguish whether the protein was in a liquid state or a glassy or fibrillar state.

Answer 1

1. To identify what proteins interact with protein-M, we can perform several experiments.
a. Coimmunoprecipitation: Use a specific antibody against protein-M and pull down all the proteins that are associated with it. Perform mass-spectrometry on immunoprecipitated proteins to know their identity.
b. Yeast-2-hybrid assay: Use protein-M as the bait and other cellular proteins as the prey to screen for proteins that interact with protein-M.4
c. Phage display
The above methods can be used identify proteins on a large scale. Once few target proteins are identified, perform Co-IP and Y2H with the mutant protein and identify how the mutation is affecting their interaction.

2. We can generate hydropathy plot for the protein and determine whether the given protein contains any membrane-spanning hydrophobic regions.
We can tag the protein with a reporter gene such as GFP/RFP and determine its cellular location by fluorescence microscopy.
We can separate membrane fraction and cytoplasmic fraction by centrifugation. Probe each fraction with antibody specific against protein-M.