Protein Structure and Function Lecture 3 Notes: Protein Folding Part 2 Assisted Folding - Some proteins fold spontaneously (e.g. RNAse), but not all proteins are capable of this (e.g. multiple domains, disulphides) and may be trapped P? 5 Unfolded 5000 5 Chaperones SO in certain unnatural/incorrect folding structures (amorphous aggregates). Chaperones Folding Energy Folding intermediates chaperones will take polypeptides out of this Partially Oligomers folded states state and help move them into the final folded states (e.g. bovine pancreatic trypsin Native state Amorphous aggregates inhibitor is assisted by enzymes in the formation of proper disulphide bonds). If Amyloid fibrils this is not possible, the protein will be Intramolecular contacts Intermolecular contacts degraded. This partially disproves Anfinsen's thermodynamic hypothesis (i.e. because protein structure is not ultimately determined by the amino acid sequence itself). - Typically, assisted folding occurs via isomerases (protein disulphide isomerase, peptidyl propyl isomerase) which help form disulphide bonds (e.g. Cyclophilin) or chaperones (make up to 30% of all cellular proteins) which deal with incorrectly or partially folded polypeptides. Chaperones facilitate the correct folding pathways by providing microenvironments so folding can occur (i.e. improving the kinetics and reducing energy barriers). - For example, unfolded bovine pancreatic trypsin inhibitor has 6 cysteine residues which are reduced and forms a major single S-S (30-51) intermediate. Next, it forms double S-S intermediates with unnatural bonds until the native double S-S intermediate is formed. Finally, the last native S-S bond is formed. However, it is possible to make this intermediate process much faster in the presence of enzymes or other molecules (e.g. B-mercaptoethanol) which break down the incorrect disulphide bonds, pushing it out of this trapped intermediate state (i.e. back into the funnel), allowing it to form correctly. - Anfinsen's experiment on RNAse uses urea and 2ME to reversibly denature ribonuclease A via reducing the disulphide bonds. The addition of urea assists the re- folding into an inactive form with randomly formed disulphide bonds. The removal of urea and addition of 2ME assists the folding into the native form (i.e. similar to role of PDI) as this allows correct formation of the disulphide bonds. Ribonuclease A folds much faster in vivo than it does in vitro. Denaturation and renaturation of RNAse Anfinsen's Experiment (1957) - urea + trace 2 ME 2 ME assists folding, play a role similar to that of PDI Native ribonuclease A + urea 2 ME -2 ME EZME Inactive ribonuclease A with randomly formed disulfide bonds - 2 ME + urea Ribonuclease A folds much faster in vivo than it does in vitro Reversibly denatured ribonuclease A; disulfide bonds have been reduced - Protein disulphide isomerase (PDI) increases the folding rate and acts as a catalyser. It helps with the rearrangement of a
polypeptide's non-native bonds via disulphide interchange and with the initial formation of disulphide bonds (formation of a mixed disulphide). - Prior to complete folding, hydrophobic fragments can be exposed to solvent, allowing for possible aggregation. This is prevented by chaperones co- and post-translationally, many induced by