a. It has been found that in some of the a-helical regions of hemerythrin, about every third or fourth amino acid residue is a hydrophobic one. Can you suggest a structural reason for this finding?

The four helices could be arranged so that the hydrophobic side chains would all point toward the center of the bundle and would pack together there. This would give a stabilizing hydrophobic core.
 

b. What would be the effect of a mutation that placed a proline residue at point A in the structure?

A proline at this position would break the helix near the Fe2 binding sites. This would probably mean that Fe2 could not be bound, and the mutant protein would be nonfunctional.
 

c. Sketch a reasonable series of steps for the folding of hemerythrin from a random coil to the structure shown.

Protein folding mechanisms are currently very controversial. Two general proposals are:

The Framework Model: In the above case, initial coiling of the chain into four consecutive helical regions, followed by the folding of these together -- perhaps aided by the binding of the Fe2.

The Hydrophobic Collapse Model: The hydrophobic effect first drives the collapse of the hydrophobic residues into a core, followed by folding of the structure into alpha helices.

2. Mutations in collagen genes result in severely crippling diseases.
 

a. Explain why mutations in the glycine residues of collagen are particularly devastating.

The small glycine residues are crucial for the formation of the triple helix of collagen, which is the basic building block of the collagen fiber.
 

b. Would you expect that collagen mutations are detrimental if only one of the two copies of a collagen gene is defective?

Yes, because a defective molecule will impair assembly of the collagen fiber even if normal collagen chains are present. This is a common property of supramolecular assemblies. Collagen mutations are therefore dominant.
 

c. The change of a glycine into another amino acid is most detrimental if it occurs toward the amino terminus of the rod-forming domain. Suggest an explanation for this.

The different severity of the mutations likely results from a polarity in the assembly process. Collagen monomers assemble into the triple-helical rod starting from their amino-terminal ends. A mutation in an "early" glycine therefore allows only short rods to form, whereas a mutation farther downstream allows for longer, more normal rods.

3. (a) When a native protein denatures it assumes a random coil, with many possible conformations.
 

(i) Given the simplest considerations, will the contribution of DS to the free energy change be + or - ?

positive
 

(ii) What requirement does this impose on DH if proteins are to be stable structures?

since DG = DH - TDS, a positive DS yields a negative contribution to DG. Thus for proteins to be stable, which requires DG to be positive for the native to random coil transition, denaturation must involve a large positive DH and/or an additional negative contribution from DS.
 

(b) Given what you know about DH and DS, explain why it is reasonable that proteins denature as the temperature is increased.

DS is positive for denaturation since order is lost, and this contributes to a more negative DG. DH is positive, but as temperature is increased, the TDS terms predominate and DG becomes more negative and the denaturation is favored.
 

(c) How might you explain why sometimes proteins denature as temperature is decreased?

The primary point here is that the solubility of hydrocarbons in water increases as temperature decreases. Therefore, there is less hydrophobic effect driving the folding.

Another possible consideration is the following -- If many hydrophobic residues are present in a protein, DS could be negative for unfolding since order may be gained in the water structure; this would result in a positive contribution to DG. But perhaps more H-bonds can form with water upon denaturation than existed in the structure, resulting in a negative DH which might lead to an overall negative DG as the T is decreased because the -TDS term gets smaller.

4. The diagram on the right is the primary sequence of a new protein that you discovered. It begins with a threonine that is at the amino terminus of the protein, and it ends with a lysine at the carboxy terminus. The sequence is to be read left to right and down, as if reading the page of a book. For each of the questions below, discuss the significance of your observations.
 

a. How many proline residues do you find? How many glycine residues? What does this suggest?

None. These are a-helix breakers and often found at turns. There may be many fewer such breaks or turns in this structure.
 

b. Color code the hydrophobic residues. Is there a repeating pattern? What does this suggest?

Yes. There is a heptad repeat with every 1st and 4th position tending to be a hydrophobic residue -- this is typical of an a-helical coiled coil. Judging from part (a), this may be an extended a-helical coiled coil.
 

c. Color code the basic residues (+ charge); then the acidic residues (- charge). Is there a repeating pattern? What does this suggest?

Yes. There are repeats of alternating positive and negative charge clusters, largely found on the outside of the predicted a-helical coiled coil. This suggests that this extented a-helical coiled coil may self-assemble into a higher order supramolecular assembly, with a stagger related to the spacing of the individual charge clusters.
 

d. Is this protein likely to be a good DNA-binding protein? Why or why not?

No. It probably has a fairly rigid, extended a-helical coiled coil structure, and it has many clusters of negative charges that will be repulsed by the DNA backbone.