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Unfolding and refolding ribonuclease

Topic: Protein structure & function

Christian Anfinsen studied bovine pancreatic ribonuclease, a 124-amino-acid enzyme that cuts RNA and is held in shape partly by four disulfide bridges. Concentrated urea (8 M) disrupts hydrogen bonds and hydrophobic interactions. β-mercaptoethanol is a reducing agent that breaks S–S bridges into free –SH groups. Removing the reducing agent and exposing the protein to air lets –SH groups oxidise and pair up into S–S bridges again. Reagents are removed by dialysis. The first four treatments follow Anfinsen's experiments; the protease treatment is an added thought experiment for comparison.

Experimental setup

Question
Does the amino-acid sequence alone contain the information needed to fold ribonuclease into its active shape?
Independent variable
Treatment applied to the purified enzyme
Dependent variable
Peptide bonds, disulfide pairing, 3-D fold and enzyme activity at the end of the treatment
Kept constant
Same purified enzyme · Same pH and temperature · Activity assayed on the same RNA substrate
Groups
  • Denatured: 8 M urea + β-mercaptoethanol, measured while both are still present
  • Renatured: Urea and β-mercaptoethanol removed together, then air oxidation
  • Scrambled: β-mercaptoethanol removed first and air-oxidised while still in 8 M urea; urea removed afterwards
  • Reshuffled: The scrambled enzyme, with urea gone, given a trace of β-mercaptoethanol for several hours
  • Digested: Denatured enzyme digested completely by a protease mixture; all reagents then removed and air oxidation allowed

Predict the state of the enzyme at the end of each treatment.

  1. Denatured: in urea + β-mercaptoethanol

    Peptide bonds intact
    Native S–S pairs
    Native 3-D fold
    Enzyme activity

  2. Renatured: both removed together

    Peptide bonds intact
    Native S–S pairs
    Native 3-D fold
    Enzyme activity

  3. Scrambled: oxidised in urea, then urea removed

    Peptide bonds intact
    Native S–S pairs
    Native 3-D fold
    Enzyme activity

  4. Scrambled + trace β-mercaptoethanol

    Peptide bonds intact
    Native S–S pairs
    Native 3-D fold
    Enzyme activity

  5. Digested by protease, then reagents removed

    Peptide bonds intact
    Native S–S pairs
    Native 3-D fold
    Enzyme activity

Answer every part to submit.