Protein Folding
Part I: Protein Folding
The shiga-like toxin (ST) is a protein-based toxin produced by the bacterium Enterohemorrhagic Escherichia coli (EHEC), which is the causative agent responsible for most cases of bloody diarrhea. Believe it or not, much of the disease symptoms associated with EHEC infection derives from the cytotoxic effects of ST, whose protein ribbon structure is shown below. Analyze these images (both the side and bottom view) and answer the following questions:
Side View Bottom View
(2 points) How many monomers (also called subunits) are in this protein? Are they similar in shape or dissimilar?
(2 points) How many individual polypeptide chains are in this protein?
(2 points) How many unique polypeptide chains do you think are in this protein?
(2 points) What type of structure is shown here (1o, 2o, 3o or 4o)?
(2 points) What would be a good descriptive (but scientific) classification for this protein? Hint: Consider our description of hemoglobin as it pertains to the nature and number of subunits contained therein.
(3 points) Identify two different forms of secondary structure in this protein (use arrows and label them appropriately). If one type of secondary structure is not present, indicate so in your answer below.
Part II: Enzymatic Active Site Motifs
Below is an image of an enzymatic active site, which is occupied by the enzyme’s substrate (shown below, left). This enzyme catalyzes the reaction that changes cyclic AMP (shown in the active site) to AMP.
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(3 points) Explain why this enzyme cannot bind to cyclic CMP and catalyze the reaction that would change cyclic CMP (shown above, right) to CMP. Be specific in your answer.
(3 points) What would happen to the enzyme’s affinity for cyclic AMP if the enzyme’s active site had a missense mutation that changed the Threonine residue (blue) to Valine? Briefly explain your answer.
(3 points) What would happen to the enzyme’s affinity for cyclic AMP if the enzyme’s active site had a missense mutation that changed the Serine residues (orange and dark green) to Threonine? Briefly explain your answer.
(3 points) Take a closer look at the active site for this enzyme and examine the noncovalent interaction mediated by the residue highlighted in pink. Is it possible for a mutation to alter this residue in such a way as to eliminate this noncovalent interaction with cyclic AMP? Briefly explain your answer.
Part III: Multimerization
Protein families arise when a protein sequence that generates a stable fold diverges over many generations and acquires new functions. One example of this can be seen in the globin family. Myoglobin (shown below, left) is a stable monomeric protein that can help carry oxygen using a heme molecule (cofactor). On the other hand, hemoglobin (shown below, right) is only functional as a tetramer and while it also uses heme to carry oxygen, it is useful over a much more dynamic range than myoglobin. The “globin fold” is structurally conserved across these proteins, but the ability to tetramerize arose through genetic drift and natural selection.
(5 points) Thinking back about what we learned about DNA sequence mutations and their effect on protein structure, provide an explanation for how changes in the polypeptide sequence of these two proteins can still produce the same overall fold (i.e. alterations that conserve protein structure) but have slight differences in the protein’s ability to multimerize (i.e. alterations that alter protein-protein interactions).
Another way to think about this is to consider what kind of mutations might promote multimerization (polar vs. nonpolar) in an aqueous environment and where would you expect these changes to be within the overall protein structure (surface vs. core)?
BONUS (1 point): Considering their structure (and what you know about synthesizing polypeptide chains), hypothesize why proline residues are often positioned at sharp turns in the polypeptide sequence. Why might that be the case? Hint: try drawing a tripeptide chain where proline is the second residue.
