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 entropy change in going from the denatured to the folded form? (c) In the native form, the molecule has four \(-\mathrm{S}-\mathrm{S}-\) 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. \((\mathrm{d})\) A gentle reducing agent converts the four \(-\mathrm{S}-\mathrm{S}-\) linkages in ribonuclease \(A\) to eight \(-S-H\) 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

In summary, the native form of ribonuclease A has lower free energy and lower entropy compared to the denatured form. The presence of four S-S linkages in the native form provides additional stability and lowers its entropy. Converting the S-S linkages to S-H bonds may affect the tertiary structure and increase the protein's entropy. Cysteine is the amino acid responsible for the formation of S-H bonds.

Step-by-step solution

a) Native form's free energy vs. denatured form's free energy

The native form represents the most stable and functional state of a protein. In comparison to the denatured form, which is an extended and non-functional chain, the native form has lower free energy. Therefore, native form has lower free energy than the denatured form.

b) Sign of the entropy change going from denatured to folded form

When a protein goes from its denatured state to the folded native form, the protein becomes more ordered and structured. The entropy, which represents the disorder or randomness in a system, decreases as a result. Hence, the entropy change from denatured form to native form is negative.

c) Effect of the four S-S linkages on the free energy and entropy of the native form

The presence of four S-S bonds in the native form provides additional stability to the protein structure, lowering the overall free energy of the native form compared to a hypothetical folded structure without the S-S linkages. In addition, the formation of these linkages imposes constraints on the protein's flexibility and results in a more ordered structure. Consequently, the entropy of the native form with S-S linkages is lower than that of the hypothetical folded structure without S-S linkages.

d) Effect of conversion of S-S linkages to S-H bonds on tertiary structure and entropy of the protein

Conversion of the S-S linkages to eight S-H bonds through the action of a gentle reducing agent would weaken the covalent linkages responsible for the stability and integrity of the native tertiary structure. The protein would lose some stability, which may lead to changes in its tertiary structure. The increased flexibility and reduced stability results in higher entropy because of an increase in the potential number of conformational states accessible to the protein.

e) Amino acid required for SH bonds to exist in ribonuclease A

Cysteine is the amino acid responsible for the formation of S-H bonds. The side chain of cysteine contains a thiol group (-SH) that can form disulfide bridges (S-S bonds) with other cysteine residues in the protein or be present as individual S-H bonds within the protein structure.

Key concepts

Free Energy in Protein Folding

Understanding the concept of free energy is crucial when studying the folding of proteins. Free energy, often denoted as Gibbs free energy (\( G \)), is a thermodynamic property that indicates the amount of work a system can perform at constant temperature and pressure. When a protein folds from its denatured state to the native state, the process is guided by the principle of free energy minimization. This means the native form of a protein is the one with the lowest free energy.

In the context of ribonuclease A, when the protein transitions from an extended, denatured form to a compact, native form, the free energy is reduced. This decrease in free energy makes the native form more stable and functionally active. The stability arises from various non-covalent interactions such as hydrogen bonds, ionic interactions, and hydrophobic packing, which collectively lower the protein's potential to do work or interact unfavorably with its surroundings.

Entropy in Protein Folding

Entropy is a measure of randomness or disorder within a system, and in protein folding, it plays a counterintuitive role. While the decrease in free energy drives the folding process, it opposes the increase in order that happens as the protein folds. A simple way to visualize this is to think of a room filled with scattered toys representing the high-entropy, denatured state of a protein. As you tidy up, the toys are organized and the room becomes ordered, analogous to the low-entropy, folded native state of the protein.

The transformation from the disordered denatured state to the ordered native structure of ribonuclease A involves a decrease in entropy. This is due to the reduced number of possible configurations the protein can adopt, much like there being fewer ways to arrange the toys neatly than to scatter them randomly.

Disulfide Bonds in Proteins

Disulfide bonds (\( -S-S- \) linkages) serve as a staple in maintaining protein structure, providing remarkable stability to the native conformation. These covalent bonds form between the sulfur atoms of cysteine residues, essentially 'locking' certain parts of the protein in place. In ribonuclease A, for instance, four of these strong bonds contribute notably to the protein's tertiary structure.

The formation of disulfide bonds lowers both the free energy and entropy of the protein. These linkages limit the mobility of the protein chain and confine it to a more ordered state, further reducing its entropy. When the \( -S-S- \) bonds in ribonuclease A are converted to \( -S-H \) bonds by a reducing agent, the protein's tertiary structure is partially disrupted, increasing its entropy due to the newly introduced flexibility.

Protein Tertiary Structure

The tertiary structure of a protein refers to its three-dimensional shape, which is critical for the protein's function. The intricate folding pattern arises from the interactions between the side chains of amino acids, leading to various bends, folds, and loops. When we discuss ribonuclease A, its tertiary structure is a globular compact shape, crucial for its enzymatic activity.

The integrity of this structure is partly maintained by disulfide bonds, but also by an array of non-covalent interactions and the overall hydrophobic effect, where non-polar amino acid residues tend to bury themselves away from water. Destabilizing the disulfide bonds, as in the reduction to \( -S-H \) groups, can perturb the tertiary structure, potentially affecting how the protein interacts with other molecules and performs its functions.

Amino Acids in Protein Structure

Amino acids are the building blocks of proteins, each with a distinct side chain that influences the protein's final structure and function. The diverse array of amino acid side chains allows for complex and specific protein structures. In the case of ribonuclease A, cysteine amino acids play a crucial role, as their side chains harbor thiol groups (\( -SH \)) that can oxidize to form disulfide bonds (\( -S-S- \)).

Cysteine's ability to form disulfide bonds adds considerable stability to the protein's structure, enhancing resistance to destructive environments. Without cysteine, ribonuclease A wouldn't possess the disulfide bonds crucial for maintaining its structure, highlighting the importance of the specific amino acid composition in dictating protein topology and resilience.