2. One could interpret the above data by arguing that some of the residues mutat
ID: 213280 • Letter: 2
Question
2. 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.
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: 115A has a substitution of alanine for isoleucine at position 15.) Protein wt I15A D16A R17A K18A F24A S25A M26A Kd (nM) 1.4 nM 1.3 nM 1.4 nM 12 nM 31 nM 1.6 nM 1.4 nM 1.5 nMExplanation / Answer
Alanine scanning is a molecular biology technique used to determine the importance/significance of a specific amino acid residue in a polypeptide to its structure or function. This is typically achieved by changing a specific amino acid to alanine and observe the effect the protein structure and function.
In this technique, alanine is used because
i. It is not bulky
ii. It is non-reactive
iii. Its methyl group mimics side chains of many of the other amino acids.
Kd is inversely proportional to binding affinity.
Greater the Kd value, lesser the affinity.
Smaller the Kd value, greater the affinity.
From the given data,
I15A: Increased the affinity
D16A and S25A: No effect on affinity
All the other mutations reduced the affinity.
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