Question

In isolation, a DNA-binding protein binds to its regulatory sequence with a Kd of 1.0 microM. Another DNA binding protein binds to another sequence on the same DNA a few bases away with a Kd of 5.0 microM when alone. The two proteins each have a domain which binds to the other, with an interaction energy of -2.7 kcal/mole: (a) Draw the thermodynamic box which represents all four states of this system. (b) what are the affinities for each protein in the presence of saturating amounts of the other?

Answer 1

kd , which represents the dissociation contant, tells us about the binding affinity of a protein.

Higher the kd value, lower will be the binding affinity. So here the second DNA binding protein has lower binding affinity as compared to the first one.

Thermodynamic cycles showing relationship between regulatory protein self-assembly and protein–DNA binding at adjacent DNA sites. Formation of the oligomeric complex bound simultaneously to both DNA sites can occur by two pathways, either separate binding of the " monomeric " protein units (dimers in this illustration ) to adjacent DNA sites followed by oligomerization on the DNA or solution protein assembly to form an oligomeric complex that binds simultaneously to both sites. The overall free energy changes for these two paths, and therefore the probabilities of complex formation , are necessarily equal.

(A) If the binding geometry of the protein complex that forms in solution does not match the disposition of adjacent DNA sites on the naked DNA, then a conformational adjustment of the protein, the DNA, or both is necessary for simultaneous binding. This will contribute to G connect in the direction to decrease the free energy change for oligomer binding as compared to separate binding to the individual sites.

(B) When the DNA sites are on separate molecules, the need for conformational adjustment is eliminated. In this case, differences between opposite sides of the cycle indicate linkage or allosteric effects mediated by noncovalent interactions across the protein oligomerization interface.

b) As said earlier , the kd values represent the binding afinity of the protein . Here the first protein have a lower kd value as compared to the second protein , therefore affinity of the first protein is much higher than the second protein.

On the other hand , affinity for the second protein will be less if the first protein is in saturation, because of the high kd value of the second protein