Question

25) The human P53 protein is a tetramer in solution, with 393 amino acids in each subunit. P53 binds to specific sequences of DNA. Imagine that you intend to use fluorescence anisotropy to investigate P53 binding to a 16 base-pair double-stranded DNA (32 nucleotides total). You have a choice of attaching a fluorophore with a high quantum yield to the P53 protein (by covalently linking to a surface cysteine), or to the DNA (using a modified nucleotide). (a) If fluorescence anisotropy is to be used to determine Kd for P53-DNA binding, would it be better to attach the fluorophore to the protein or the DNA? Explain your choice. (4 pts) (b) A fluorescence anistropy experiment indicates that the DNA and P53 bind with a Kd of 25 nM+ 5 nM. The same protein-DNA interaction was investigated using SPR (with P53 immobilized by its N- terminus), and the value of Kd was found to be 420 nM + 50 nM. Assuming each experiment was performed correctly with properly functioning instruments), provide an explanation for the difference between the fluorescence anisotropy and SPR results. (4 pts)

Answer 1

(a) Given that the fluorophore has a high quantum yield and fluorescence anisotropy is used to determine Kd for protein-DNA interaction, the quantum efficiency or the probability of an energy-transfer event occurring will depend on the Forster distance (R0) as

$E = \dfrac{1}{1+\bigg(\dfrac{r}{R_0}\bigg)^6}$

Thus, smaller the distance of the fluorophore for a given biomolecule, bigger will be the quantum yield to detect protein-DNA binding. Thus attachment of the fluorophore to the smaller biomolecule (in this case, DNA) will favor greater observation of the binding event, than the bigger biomolecule (in this case, p53 protein).

(b). Due to its bigger size, p53 protein takes greater space while interacting with the wandering DNA oligonucleotides. Thus for a single tetrameric protein immobilized on a surface, multiple DNA molecules can bind at a time, thus, the concentration of the biomolecules will be greater due to 1:4 (or probably more) interaction with the protein that produces SPR. This would also have a greater variation in values due to the same reason. On the other hand, fluorescence anisotropy is a 1:1 interaction where binding is observed once fluorescence is noted at very small distances. This can occur at much lower concentrations and with less variations.