Question

Problem 7 (6 points): Christian Anfinsen made a famous experiment in which he completely unfolded, and re-folded the enzyme RNase A using urea and beta-mercaptoethanol (BME). Explain how the two molecules urea and BME affected the unfolding and refolding of the enzyme. What were the two important conclusions from this experiment?

Answer 1

Answer: 7

Answer: In the year 1950, a series of experiments were conducted by the Christian Anfinsen which demonstrates that the information needed for the formation of 3-dimensional actives proteins which lies within its amino acid sequence. Further experiments generalized this idea that the conformation of the protein is determines by its primary structures.

He performs his experiments with a particular type of enzyme (Ribonuclease) of 124 amino acid sequence, in tertiary structure with four disulfide bonds

Function of Denaturing agents

o useq - . ll C - NH₂ ZA - disrupts the non-covalent Intersctions (Ionic mteraction and Hydrogen bonds) B-Mercapto ethanol sh - Vig the an oxidation - reduction it breaks down disulfide bonds

Reactions demonstrating the function of BME and Urea

Reaction 1:

On treatment with excess BME and Urea, the native enzyme (enzymatically active) loses its hydrogen and disulfide bonds. When these agents are removed enzymes regains its original tertiary structure.

H₂N, Exce Bме Dobago Oot 8.0M use a COLOCOS ceo00 Native form 0- denatured enzyme When agent Removed.

Reaction 2: When BME removed first, it results in the formation of the scrambled structure of the enzyme (the inactive enzyme) because of improper disulfide bond formation.

2.1 ce & BME Removal 0000 doooo ( X doo وار 800000 පලතුරම මත. e uses PY1OY * * 8 ooooooooo C=0 0- scrambled enzyme Denatured enzyme

Reaction 3: When the scambled enzyme formed during reaction 2 treated with the trace amount of BME it gains its correct tertiary structure (enzymatically active native form).

000 Pogado Go Trace amount of BME Scrambled enzyme Native form

Conclusion: When BME and Urea was removed a spontaneous refolding of protein was observed. When Urea removed first protein would be replicated efficiently, while on the first removal of BME the protein could not replicated efficiently and results in scrambled form, which on treatment with the trace amount BME restores the tertiary structure of the enzyme.

Thus we can conclude that the catalytic activity and 3D structure of ribonuclease is lies within its amino acid sequence.