6. Lysozyme is an enzyme that hydrolyzes sugar linkages in the bacterial cell wall and was first discovered by Alexander Fleming. It is widely used throughout the food industry as a preservative to prevent food spoilage. To improve its stability, disulfides were introduced into lysozyme. Two variants were made, one with a disulfide between residues 3 and 97, and a second with a disulfide between residues 21 and 142. Both have increased stability, with ΔTm = +7 °C (Cys3-Cys97) and ΔTm = +10 °C (Cys21-Cys142). Explain why the thermal stability increases. Assuming both disulfides have the same bond strength, explain why the Cys21-Cys142 has a higher Tm than the Cys3-Cys97 variant. Hint: ΔTm is the temperature at which half the protein is in its unfolded state. Think in terms of the entropy difference between the native and mutants and what that does to the free energy state of the protein (6 points).

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Explanation:

The Cys3-cys97 and cys21-cys142 disulfides restrict the unfolded state of lysozyme enzyme to a class of more compact structures with a less exposed hydrophobic surface, compared to the unfolded states of reduced/non-crosslinked lysozyme. there are 2 major factors which lead to the stabilization of lysozyme due to disulfide bonds-

1- increase in the loop size due to the formation of disulfide bonds that leads to an increase in the even entropic effect.

2- the region formed should be flexible. the strain energy due to the formation of the disulfide bond is lower.

cys21-cys142 has a higher Tm than the cys3-cys97 because it involves flexible parts of the molecule. 21 and 142 residues are located on opposite sides of the active-site cleft where significant hinge-bending motion is seen. this introduces minimal strain in the protein.

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