Protein folding

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Refolding proteins without chaperones

  • The denatured protein must first be diluted in a buffer and stirred.
  • If the buffer volume is large and there aren't many exposed hydrophobic residues, there is a good recovery rate.
  • If the buffer volume is small and there are a lot of exposed hydrophobic residues, there is a bad recovery rate.
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Chaperone action

  • Chaperones usually bind exposed hydrophobic residues, reducing the concentration of exposed hydrophobic residues and their irreversible aggregation.
  • They undergo cycles of binding and releasing, allowing local regions to fold, which are then no longer bound by the chaperone.
  • Eventually the protein folds by a specific pathway. 
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An example of a prokaryotic chaperone: trigger fac

  • aka bacterial ribosome associated chaperone.
  • Has 3 domains: ribosome binding; substrate binding; and peptidylpropyl isomerase. 
  • Has 4 hydrophobic patched, which bind to the exposed hydrophobic residues of the protein.
  • The RBD binds to protein L23 of the ribosome. 
  • Fast on/off rates leads to localised folding.
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Two examples of eukaryotic chaperone systems.

  • Nascent associated complex - similar to trigger factor
  • Ribosome associated complex - Hsp80/Hsp40. 
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E.Coli post translational secretion

Done if the signal sequence is not hydrophobic enough

1. The protein is bound by SecB, which prevents folding. 

2. Then, the protein is delivered to SecA, which is an ATPase. 

3. ATP hydrolyses allows sec A to act as a ratchet, forcing the peptide through the pore, which is called secYEG.

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Co-translational translocation

1. A signal recognition protein (SRP) binds to the SRP receptor and the signal sequence.

2. Resultantly, the ribosome is moved in line with the transporter. 

3. Translation resumes, and the protein is forced through the membrane.

4. The peptidase Spase cleaves the signal sequence. 

 

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Eukaryotic ER import

1. Oligosaccharyltransferasee glucosidase I and II glycosylates the first asparigine that crosses to the luminal side through sec61(alpha beta gamma).

2. A Hsp70 system acts as a ratchet composed of:

                           The J domain protein sec63 in a complex with sec61. 

                           This stimulated the ATP ase activity of Hsp70 (bip).

                           Bip pulls in the nascent polypeptide as a result of the two being stimulated                                       to bind by ErdJ.

3. The glycoslyated asparagine is recognised by calnexin and calreticulin after the nucleotide exchange factor bap causes bip to disengage from the protein. They are very simuler but calnexin is membrane bound. 

4. The two chaperones recognise Erp57, which forms and breaks disulphide bonds, allowng correct disulphide bond formation

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