You have a generated an RNAse A mutant in which 2 of the 8 cysteines have been m

Question

You have a generated an RNAse A mutant in which 2 of the 8 cysteines have been mutated to alanine. Despite these mutations, the protein retains 100% activity. You now subject the protein to treatment with 8 M urea and mercaptoethanol What percent activity will you expect the protein to have in 5. each of the cases below? You first remove the -mercaptoethanol by 12 hours of dialysis and then remove the urea by 12 hours of dialysis. a. b. You remove the -mercaptoethanol from your solution by dialysis over a period of 12 hours. You first remove the urea by 12 hours of dialysis and then remove the - mercaptoethanol by 12 hours of c. dialysis. You simultaneously remove the urea and -mercaptoethanol from your solution over a period of 12 hours by dialysis. d.

Explanation / Answer

urea - break down non-covalent bonds such as hydrogen bonds holding the secondary structure.

beta-mercaptoethanol - to reduce and break down the disulfide bonds in the tertiary structure.

A. protein refolded but no longer biologically active. Because the presence of urea inhibit the formation of non covalent bond it leads to disulfide bonds formed incorrectly.

B. No functional protein

C. protein refolded back into its original biologically active form. beta mercaptoethanol was enough to catalyze the breaking of the incorrect disulfide bonds. Eventually the protein formed the correct disulfide bridges and returned to its native form.

D. protein refolded back into its original biologically active form.

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