Discovery Talk Questions and Answers: Chaperonin-assisted Protein Folding

Arthur Horwich

Assessments created by Dr. Nathan H. Joh.

Questions

Select the answer choice with the most appropriate words to fill the blanks below.
Anfinsen discovered in 1961 that the four disulfide bridges in ribonuclease, which were reduced by denaturing the protein in 8 M urea in the presence of beta-mercaptoethanol, formed again and the protein regained activity upon dialysis followed by air oxidation. This indicates that the protein ___(a)___ correctly; and implies that the information necessary for achieving correct three-dimensional structures of proteins is contained within the ____(b)____ sequence. [Video]
1. (a) recombined, (b) DNA
2. (a) spliced, (b) RNA
3. (a) folded, (b) amino acid
4. (a) oxidized, (b) cysteine
Consistent with the hydrophobic effect, correctly folded proteins generally pack their hydrophobic residues within the protein core, while exposing the hydrophilic residues on the surface. This would allow hydration of individual protein molecules, thereby resulting in their solubilization.
Knowing this, explain why misfolded proteins often result in aggregation (e.g. formation of inclusion bodies in E. coli).
According to Dr. Horwich’s hypothesis, why would the ornithine transcarbamylase (OTC) expressed as a full-length protein in a certain yeast mutant lack its native biological activity?
Select the best explanation(s) from the list below.
1. OTC folded improperly due to the loss of information necessary for achieving the protein’s correct three-dimensional structure.
2. OTC folded properly due to the retention of information necessary for achieving the protein’s correct three-dimensional structure.
3. OTC folded improperly due to the loss of cellular factors that assist in proper OTC folding.
4. OTC failed to migrate into the mitochondrial matrix.
5. None of the above
Dr. Horwich discovered that yeast protein Hsp60 chaperoned the correct folding for multiple proteins, and that, as a result, the protein class earned its name, “chaperonin”. The crystal structure for the bacterial homolog to chaperonin, groEL, reveals that the hydrophobic surface is exposed inside the open ring of groEL complex.
What is the common structural feature of non-native proteins that enables the interior of the open ring of the chaperonin to interact with them?

Briefly explain.
Which of the following statements best describe(s) the mechanism by which correct protein folding takes place once the misfolded protein binds the open ring of groEL? Select all that apply.
1. The groEL ring closes to isolate the protein and provide steric hindrance that mechanically refolds the protein.
2. The groEL ring closes to provide a hydrophilic space to isolate the protein and inhibit its aggregation with others until properly folded.
3. The groEL ring closes to isolate the protein and decode the information necessary to achieve the correct three-dimensional structure.
4. The groEL ring closes to isolate the protein and provide enough time for the protein to properly fold on its own.
5. None of the above
Dr. Horwich and coworkers determined that Hsp60 was the gene required for further processing (chaperonin of folding) of polypeptides entering the mitochondrial matrix space. “Hsp” comes from an acronym for “heat-shock protein”, as yeast expresses the products of this group in larger quantity when heat shocked. Where does “60” in the name of the gene come from?
Dr. Horwich and coworkers observed that treating mitochondrial suspensions with chloroform resulted in the assembled β-subunit of F1-ATPase (F1β) to partition into aqueous phase, whereas non-assembled F1β partitioned into the organic phase.
What does this indicate? (Select all that apply)
1. F1β assembles only when folded properly.
2. The non-polar core of F1β is exposed when it is misfolded and interacts with non-polar organic solvents.
3. Misfolded F1β do not properly assemble.
4. None of the above
To test whether the blockage that disabled α143 mutant yeast to chaperone the folding of mitochondrial proteins was before or after import of precursor proteins into mitochondria, Dr. Horwich and coworkers incubated radioactive [35S] methionine-labeled precursor proteins with isolated mitochondria, followed by proteinase K digestion. Then, the size and quantity of radioactive contents of mitochondria were analyzed on SDS polyacrylamide gel electrophoresis (SDS-PAGE) coupled with fluorography.
For the experiment performed with wild-type mitochondria, only the mature-sized forms of tested proteins were resistant to proteinase K digestion, while α143 mitochondria resulted in all forms (precursor-, intermediate- and mature-sized forms) to be resistant to proteolysis.
1. In each cases what provided the protection to proteolysis?
2. b. Taken together, do these results indicate that the inhibition of chaperon activity takes place before or after import of precursors into mitochondria?

Answers

Answer c is the most relevant.
Aggregation takes place due to non-specific intermolecular interaction among protein molecules. Hydrophobic residues packed within the protein core in the context of natively folded state would be exposed to surface upon misfolding. Once exposed, hydrophobic residues from one misfolded protein molecule may interact with exposed hydrophobic residues from another, forming non-specific intermolecular interactions, thereby leading to aggregation.
c.
Non-native proteins would expose hydrophobic residues that would have been packed within the core of the protein in the native context. These exposed hydrophobic residues can then nonspecifically interact with the hydrophobic patch within the open ring of groEL.
b and d.
60 corresponds to the protein’s molecular mass of 60kD.
a, b and c
1. The process of importing proteins into the mitochondria provided the resistance against proteolysis.
2. These results indicate that the blockage that disabled the chaperone activity takes place after import into the mitochondria.