Question

The protein ribonuclease A in its native, or most stable, form is folded into a compact globular shape: (a) Does the native form have a lower or higher free energy than the denatured form, in which the protein is an extended chain? (b) What is the sign of the system's entropy change in going from the denatured to the folded form? (c) In the native form, the molecule hasfour \(-\mathrm{S}-\mathrm{S}-\) bonds that bridge parts of the chain. What effect do you predict these four linkages to have on the free energy and entropy of the native form relative to the free energy and entropy of a hypothetical folded structure that does not have any \(-\mathrm{S}-\mathrm{S}-\) linkages? Explain. (d) A gentle reducing agent converts the four \(-\mathrm{S}-\mathrm{S}-\) linkages in ribonuclease \(\mathrm{A}\) to eight \(-\mathrm{S}-\mathrm{H}\) bonds. What effect do you predict this conversion to have on the tertiary structure and entropy of the protein? (e) Which amino acid must be present for \(-\mathrm{SH}\) bonds to exist in ribonuclease \(\mathrm{A}\) ?

Short answer

(a) The native form of ribonuclease A has lower free energy than the denatured form. (b) The sign of the system's entropy change in going from the denatured to the folded form is negative. (c) The four disulfide bonds in the native form lower its free energy and entropy compared to a hypothetical structure without them. (d) Converting the disulfide linkages to eight -SH bonds would weaken the protein's tertiary structure and increase its entropy. (e) The amino acid responsible for the presence of -SH bonds in ribonuclease A is cysteine.

Step-by-step solution

(a) Native vs. Denatured Free Energy

In general, the native form of a protein has a lower free energy than the denatured form, as the native form represents the most stable conformation, which usually has lowest available free energy. Therefore, the native form has lower free energy than the denatured form.

(b) Entropy Change Sign

Entropy is related to the randomness or disorder of a system. In the case of a protein, a denatured protein is an extended chain with more possible conformations, which means a higher entropy. Therefore, when transitioning from the denatured to the folded (native) form, the entropy of the system decreases, and the sign of the entropy change would be negative.

(c) Disulfide Bonds Effect on Free Energy and Entropy

Disulfide bonds (\(-\mathrm{S}-\mathrm{S}-\)) contribute to the stability of the native form of a protein by forming covalent bonds between specific cysteine residues. These bonds reduce the number of possible conformations the protein can take, thus decreasing the entropy of the native form. However, these bonds contribute to the lowering of the system's free energy since they play a role in the structural stability of the protein. Comparing with a hypothetical folded structure without these linkages, the native form with disulfide bonds has lower free energy and entropy.

(d) Reducing Agent Effect on Tertiary Structure and Entropy

A gentle reducing agent converts the four \(-\mathrm{S}-\mathrm{S}-\) linkages in ribonuclease A to eight \(-\mathrm{S}-\mathrm{H}\) bonds. This conversion would weaken the stabilizing forces and the tertiary structure of the protein, leading to an increase in entropy due to increased conformational possibilities. At the same time, the free energy of the protein may increase.

(e) Amino Acid for \(-\mathrm{SH}\) Bonds

The amino acid residue responsible for the presence of \(-\mathrm{SH}\) bonds in ribonuclease A is cysteine. Each cysteine residue contains a thiol group (-SH), which is involved in the formation of disulfide bonds between two cysteine residues.

Key concepts

Free Energy

Free energy is a key concept in understanding why proteins fold into their native structures. In biological terms, proteins seek a low energy state to stabilize their structure. The native form of a protein, such as ribonuclease A, is more stable and compact than its denatured, or unfolded, form. This stability is directly related to free energy.
In a denatured state, the protein has an extended shape and higher energy due to greater instability. As proteins fold into their native structures, free energy decreases. Lower free energy means the protein structure is more stable.

• Native form = lower free energy (more stable)
• Denatured form = higher free energy (less stable)

This relationship is crucial for understanding protein behavior in different environments.

Entropy Change

Entropy deals with disorder or randomness in a system. For proteins, the unfolded form offers more configurations and thus, higher entropy. Transitioning from the denatured to the folded form reduces this randomness, leading to a decrease in entropy.
In the folding process of ribonuclease A, the entropy change is negative as the protein becomes more ordered. This is because:

• Unfolded form (denatured) = higher entropy (more disorder)
• Folded form (native) = lower entropy (less disorder)

Understanding entropy change helps explain why proteins naturally fold under physiological conditions.

Disulfide Bonds

Disulfide bonds are powerful covalent bonds that play a vital role in protein structure. They form linkages between specific cysteine residues. In proteins like ribonuclease A, these bonds stabilize the native structure.

By limiting the protein's conformations, these bonds lower entropy and enhance stability. This means that a protein with disulfide bonds will have:

• Lower entropy compared to hypothetical non-bond structures
• Lower free energy due to increased structural stability

The presence of disulfide bonds is a critical factor in maintaining the structural integrity of proteins.

Cysteine Residues

Cysteine is the amino acid responsible for the formation of disulfide bonds due to its thiol group (-SH). These bonds play a pivotal role in the tertiary structure of proteins like ribonuclease A.

When a reducing agent converts disulfide bonds ( (-S-S-) ) to thiol groups ( (-S-H) ), the stability and structure of the protein weaken. This conversion increases entropy and can lead to a higher free energy state. Having cysteine residues ensures that these essential links can form, contributing to protein folding by:

• Providing sites for disulfide bond formation
• Enhancing structural stability and lowering free energy

Cysteine residues are thus integral to maintaining the desired protein conformation.