Protein Z is a transcription factor that binds DNA. The interaction of protein Z

Question

Protein Z is a transcription factor that binds DNA. The interaction of protein Z with DNA was analyzed via alanine scanning. The following table reports binding data for the wild-type protein (wt) and for various mutant proteins. (Nomenclature: I15A has a substitution of alanine for isoleucine at position 15.)

                                    Protein             Kd (nM)

                                    wt                    1.4 nM

                                    I15A                1.3 nM

                                    D16A              1.4 nM

                                    R17A               12 nM

                                    K18A              31 nM

                                    F24A               1.6 nM

                                    S25A               1.4 nM

                                    M26A              1.5 nM

Which two mutations affect the Protein Z-DNA interaction to the greatest extent? Do these mutations increase or decrease binding affinity? Justify your answers.

One could interpret the above data by arguing that some of the residues mutated in the study are directly involved in binding DNA. (They interact with the DNA.) In a few sentences, support this idea by discussing the principle behind an alanine scanning experiment. Make sure to explain why we use alanine as the replacement residue in these experiments.

Suppose that additional experiments with Protein Z demonstrate that the residues mutated in this study do not, in fact, contact DNA. Given these observations, suggest an alternate explanation for the binding data with the mutant proteins.

Explanation / Answer

In this protein two mutations R17A showing Kd value 12nM and mutation K18A having kd value 31 nM. That is quite high as compare to wild type 1.4 nM. These two mutations decrease the binding affinity.

DNA is negatively charged due to presence of phosphate group and it interacts with the positively charged amino acids. Hence arginine and lysine may play the crucial role. As alanine is non bulky, inert methyl group and without charge containing amino acid can be use for the best to examine the role of any amino acid especially charged acidic or basic residues. Hence, this amino acid is considered best for the mutation analysis.

As find that these residues are directly interact with DNA than it is also possible that these amino acids playing crucial role in the maintaining the structure of the enzyme that it interact with the DNA. Therefore once the structure of DNA has lost hence they showed the increases binding constants. It can be further validated by analyzing crystal structure or NMR studies.

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