2. The wild-type molecular chaperone GroEL consists of two stacked rings of seve

Question

2. The wild-type molecular chaperone GroEL consists of two stacked rings of seven subunits with a central cavity within which protein folding is assisted. A mutation form of GroEL, K3E (lysine-3 has been replaced with a glutamic acid) reduces the subunit interactions so that the mutant is monomeric, and shows no chaperone activity. Circular dichroism (CD) was used to monitor the binding of substrate (ATP) to both GroEL K3E (Fig. A) and wild-type GroEL (Fig. B). In both Figs. A and B, the thick lines represent the CD spectra of the ATP-bound proteins, and the thin lines represent the CD spectra of the apoproteins. (a) Briefly comment on the effect of ATP on the secondary structure of GroEL K3E based on the CD spectra in Fig. A. (b) Briefly comment on the effect of ATP on the secondary structures of wild-type GroEL based on the CD spectra in Fig. B. (c) A mutation study shows that the oligomeric states of the wild-type and mutant GroELs follow a rank order of decreasing stability: wild-type > K3R> K3I> K3E. Based on the result of the mutation study, suggest a possible role of lysine-3 in the assembly of the quaternary structure of GroEL 8 205 225 245265 Wavelength, nm

Explanation / Answer

Ans A- GroEL K3E mutant shows difference in CD spectra of apoproteins and ATP bound proteins, this can be due to the absence of lysine residue ATP are not able to bind with apoproteins as the binding site key amino acid interaction is absent in GroEL K3E. This happens due to replacement of basic amino acid lysine with an acidic one glutamate.

Ans B- lysine being a basic amino acid seems to play a key role in binding of ATP to the apoprotein subunits because as shown by the CD spectra of wild type GroEL the graph of ATP bound proteins and apoproteins have same values.

Hence it clearly shows the role of ATP in stacking of two rings of seven subunits and involvement of lysine for interaction with ATP.

Ans C- As shown by the mutation study the assembly of groEL is affected by the change in charge of amino acid present at residue three of the active site. This key residue results in dimerization and interaction of the GroEL with ATP. As the charge becomes positive the protein subunits are unable to interact to form quarternary structure. Hence when lysine is replaced by arginine another basic amino acid the assembly is partially lost, and when it is replaced by Isoleucine a neutral amino acid subunit assembly is affected more due to loss of negative charge. When negative charged lysine is replaced by positively charged glutamate the assembly function is totally lost because electrostatic interactive force between the subunits are now acting as electrostatic repulsive forces.

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