You have isolated a sequence-specific DNA-binding protein (Protein X) with the f

Question

You have isolated a sequence-specific DNA-binding protein (Protein X) with the following primary sequence: This protein binds as a homodimer, and sequence homology studies with other proteins from the same family revealed that in all other related proteins, residue 61 is an arginine (as opposed to a phenylalanine). In the Protein X F61R mutant, sequence-specific binding is lost. The Protein X F61W mutant was also made and more studies carried out. (a) Why generate a F61W mutant? (b) FRET experiments of the binding of wild-type protein to different DNAs (with different absorption characteristics) demonstrated that while energy transfer between Protein X and DNA was occurring, it was not the only mechanism of excited-state deactivation of the bound protein. In the case of the F61W mutant, the donor and acceptor groups were spatially closer together, but other excited-state deactivation mechanisms were more efficient than in the wild-type protein. Remember that the equation that describes the energy transfer as a function of spectral overlap is: Q_r/Q_k = Gamma + k_s/Gamma + k_f + K^2 Q_f/r^6 n^6 (8.71 times 10^23 s^-1) f On a simple graph, plot the expected energy transfer curves for both cases. Does this necessarily mean that the two proteins have different modes of binding? Explain.

Explanation / Answer

Why generate a F61W mutant:

F61 W is the strain which is wild type for phenylalanine at 61 position. The other which has substitution of Arginine (R) is given as F61R mutant. This is needed to make mutant of both strains for observing the changes in protein X functions. After making mutant both will has loss of amino acid at 61 positions, then both strains’ protein X will become same in composition of amino acids. Now the corresponding characteristics will be observed.

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