Methods of studying Protein Interactions
What is similar about Affinity chromatography and Pull down assays
Both processes involve "Fishing". Affinity chromatography is fishing for your POI with another substance (like nickle) as bait. Pull down assays is like fishing for a protein with another protein as bait to see if they interact.
What is a stress granule
Membraneless pockets of cell material that forms during times of stress. (housekeeping proteins are held in storage in the stess granule while chaperone and antixodants are formed to deal with the stress)
What does a Red shift indicate in Trp fluorescence?
the protein containing the Trp underwent a conformational change that exposed the Trp to a more polar environment
What is being diffracted during CRYO-EM
Electrons are diffracting is a specific pattern when they hit the Cryo sample
What distinguishes CO-IP under the umbrella of pull-down assays
Centrifugation
What are intrinsically disordered proteins
proteins with no distinct structure because polypeptide chains are constantly switching structures. They are really important in coupling folded domains in proteins.
Define chromophores and fluorophores, give one example of each.
Chromophore: have functional groups that absorb UV light. Ex. aromatic rings, peptide bonds or cystines
Fluorophore: have functional groups that absorb and emit UV light. Ex. Trp
What does it mean to "walk the amino acid" and why is it necessary.
walking the amino acid means determining the specific amino acid at a peake in 2D NMR spectorscopy.
If little light is passing through your sample in Biolayer interferometry, what can you infer about protein interactions?
There is much interaction between target protein and antibodies, blocking light from reaching the SA biosensor.
What are excited states "Invisible"
They have shorter lifetimes, so its difficult to gather a signal before the molecule returns to the ground state.
What is chemical crosslinking?
Freeze capturing a protein interaction by forming covalent bonds between functional groups to stabalize the partners.
what information are you getting out of X-Ray crystallography that you can use to determine protein structure
The diffraction pattern of the X-ray can tell you the angles of phi and psi in the polypeptide backbone.
Explain why you may observe emissions of different wavelengths than that of your fusion protein during FRET.
How does an MRI distingusih form cancer and healthy tissue
Metabolites in cancerous tissue exchange protons with the water when we excite the hydrogen associated with the metabolites. This will change the energy and temp of the water. In healthy tissue there are no metabolites so the lack of exchange of protons means the energy and temperature of the water remains constant.
What is a common issue with chemical crosslinking?
It can generate false positives by gluing together proteins that don't naturally interact with each-other.
Waht are the 3 example of nuclei that can be used in NMR spectroscopy?
H, C13, and N15
Explain how BioID allows identification of protein-protein interactions.
A protein tag, BirA, attaches to your POI forming a fusion protein. BirA will biotinylate and proteins that the fusion protiens comes near. Biotinylated proteins can then be purified to see which proteins the POI likely interacts with.
Describe what generates a peak in NMR
The peak comes from the energy that is release when the nuclei go from excited state back to their group state after the pulse excited them. The energy released is equal to the energy of the pulse that was given to it.
What is an example of a chemical crosslinker
Formaldehyde
what are the characteristic CD spectra of alpha helix, beta sheets and random coils.
alpha helix: 222(-) 208(-)195(+)
Beta sheets: 217 (-)
Random coil: 199 (-)