Question

In the Anfinsen protein folding experiment the sequence of removing urea or BME makes a difference for functional refolding of the protein. Explain why removing BME first leads to a nonfunctional enzyme, but removing urea first leads to a functional enzyme. Why does adding small amounts of BME alone to the solution with the nonfunctional enzyme "rescue" the function

biochemistry

Answer 1

Protein folding is driven by multiple forces. One of these is the hydrophobic effect, which in order to reduce the total entropy of the system, will drive the protein's hydrophobic residues (such as valine and leucine) to cluster in the inner core of the protein away from the aqueous solution. This clustering of hydrophobic residues in the inside of the protein helps the protein to fold correctly. Most denaturants like urea and guanidine hydrochloride will reduce the entropic gain that results from this tendency--this results in denaturation of the protein. 2-ME, on the other hand, doesn't lead to denaturation in this way, instead, it just reduces disulfide bonds. By adding a denaturant like urea, and a reducing agent, like 2-ME, you are causing the protein to become unfolded in two distinct ways. Also, I believe that another key part of this experiment is that in order to allow the protein to re-fold correctly, you are only adding TRACE amounts of 2-ME--thus disrupting all but the most energetically favorable disulfide bonds that are present in the native conformation. Other, less favorable disulfide bonds will be disrupted by the low concentration of 2-ME

In the presence of urea the protein was unfolded and it could adopt any number of conformations. In some of these random conformations there were two cysteine residues that were close together and a disulfide bridge formed between them. This locked the polypeptide into a misfolded structure with no activity.

If you remove the urea, the protein folds rapidly into the proper three-dimensional structure. The "correct" disulfide bridges then form between the cysteine residues that are adjacent in the correctly folded structure. This locks in the three-dimensional structure and prevents it from unfolding in the harsh conditions outside the cell.