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Could you help explain each problem from this document on courshero?

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Project Part 3
You are setting up a competition assay for your target protein. You plan to identify potential
inhibitors for your protein by screening a library (i.e., individually testing a large number) of
compounds. These inhibitors will not be great binders, but the identification of a scaffold that
acts as an inhibitor can generate a "lead compound." Medicinal and organic chemists can then
optimize the drug from the lead compound to generate a molecule with great affinity for your
protein.
You plan to use a fluorescence anisotropy based assay, called competition assay, where your
protein will bind a peptide that has a fluorophore attached. If one of the tested compounds acts as
a competitive inhibitor, there will be fewer peptides bound to your protein, which would lead to a
change in anisotropy.
In your assay, you plan to use 10 nM of your kinase, and 10 nM of your fluorophore-labeled
peptide. You have three peptides that you could use for your screening. Peptide A has a Ka with
your kinase of 100 pM, and peptide B has a Ka with your kinase of 10 nM, and peptide C has a
Ka with your kinase of 1pM.
For initial drug screen, the binding of inhibitor to protein is usually not very strong, with Ka
around 1 pM.

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1) Would you expect the measured anisotropy signal to increase or decrease when you add an
inhibitor, which lowers the amount of protein-peptide complex? (Hint: Look at the "r" equation
that describes anisotropy. Here you are measuring the intensity of fluorescent light that is parallel
and perpendicular to the polarized light that you added to solution. If a molecule is completely
stationary, the measured fluorescence would be only parallel, with no perpendicular intensity. If a
molecule is spinning rapidly, the measured fluorescence would have equal amounts of parallel
and perpendicular intensity.) (5 points)
r=
In + 21

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2) Calculate the percentage of protein-peptide complex if you have no added inhibitor and you
use peptide A, B and C. (Hint: we gave all the numbers you need above. Be careful to note how
the protein concentration compares with the ligand concentration and the Ka value.) (10 points)

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