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In: Biology

Imagine conducting the following experiment. Take a sample of the native folded peptide and measure the...

Imagine conducting the following experiment. Take a sample of the native folded peptide and measure the amount of peptide required to inhibit the activity of a potassium channel, say the Kv4.2 channel, by 50%. We will call this value the IC50 value. For our peptide, IC50 = 5 nM for the Kv4.2 channel. Treat the peptide with a mixture of 2-mercaptoethanol and guanidine hydrochloride. Remove the 2-mercatoethanol and then the guanidine hydrochloride to allow the peptide to refold. Treat the Kv4.2 channel with the refolded peptide and measure the IC50 value. If the IC50 value is now 250 nM, does this peptide sequence obey the Anfinsen dogma? Explain.

Solutions

Expert Solution

In this case, the peptide are treated with a mixture of 2-mercaptoethanol (2 ME) and guanidine hydrochloride. But while refolding, 2-mercaptoethanol is removed first then the guanidine hydrochloride is removed. We know that, 2-mercaptoethanol is used to disrupt the disulfide bonds present in the peptide, Whereas, urea or in this case, guanidine hydrochloride is used to denature it. In Anfinsen's experiment, he couldn't denature ribonuclease A only with 2 ME so he added urea. The protein then unfolded and when he removed 2 ME and urea, the protein refolded and gained functional activity. But when he removed 2ME but not urea, only 1% of functional activity recovered. So, he finally removed 2 ME first, then urea but again added a little amount of 2 ME. In the presence of only urea, the proteins are still unfolded and it could adopt any random conformation. Protein folding is occurred by many forces like hydrophobic forces also. Denaturants like guanidine hydrochloride or urea reduce the entropy and protein denatures. But, 2 ME only breaks the disulfide bonds. So protein is actually unfolding by two different mechanisms by using 2 ME and guanidine hydrochloride. So the reason behind adding trace amount of 2 ME to refold the protein correctly, so that it can disrupt all but the most energetically favorable disulfide bonds that are present in native protein or peptide form. And, other less favorable ones will be disrupted by a low level of 2 ME, giving the perfect refolded protein or peptide.

In this present experiment, while removing urea, no trace amount of 2 ME is added to obtain a perfect native folded form of the peptide. So, may be the peptide did not fold with exact strong disulfide bonds but may have some random disulfide bonds. So the activity of the peptide is reduced and the amount required to inhibit 50% of potassium channels, i. e., IC50 increased from 5nM to 250nM. But as this experiment doesn't completely follow Anfinsen's experiment, so it cant be commented whether this obeys Anfinsen's dogma or not. But, if Anfinsen's dogma would be obeyed then the peptide activity would remain the same.


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