AnfinsenexperimentDisulfidebridges can be disrupted by treating a protein with 2-metaptoethanol.
The bondbetween two sulfurs can be broken and a new bond can be created between the twosulfurs at the end of the two molecules of 2-merapthoethanol. The aim ofAnfinsen’s experiment was to show that the information for protein foldingresided entirely within the amino acid sequence of a protein (the primary structure).A ribonuclease A was used in the experiment and denatured the protein with denaturanturea and 2-metaptoethanol.
Urea is a chaotropic agent which lets water moleculesto solvate non-polar groups inside the protein which disrupts the hydrophobic interactions.Theprotein then unfolded when this was added. It lost is original conformation andbecome further inactive due to the enzyme’s active site changing.
It refolds spontaneously and regains biologicalactivity when you remove the urea and 2ME from the solution. Removingthe 2-metapoethanol but not urea led to recovery of 1% of the activity. This isdue to the random sulfide bridges between the 8 cysteines present in the protein.
Recovery is no 100% which lead to the discovery of the enzyme: protein disulfideisomerase (PDI)- an enzyme that catalyzes reduction of incorrect disulfidebonds and allows a protein trapped in an incorrect conformation to unfold andtry again. Reference:http://sandwalk.blogspot.co.uk/2007/02/anfinsen-experiment-in-protein-folding.
html Thediagram above shows the overall steps of the Anfinsen experiment, it shows thenative ribonuclease A being added to urea and 2ME which is then denatured andthe disulfide bonds have been reduced. Biosynthesisof insulin Insulinis a 51 residue protein that is composed of two peptide chains linked by twodisulfide bonds. It plays a big role in the regulation of human metabolism. Insulinis synthesized in rough endoplasmic reticulum in the beta cells which arelocated in the pancreas. There are different types of insulin, there is rapidacting, short acting and long acting.
The structure of it determines how easyit can fold, trafficking, self-assembly and receptor binding. https://www.ncbi.nlm.nih.gov/books/NBK279029/ It is synthesized by precursor cells whichinclude preproinsulin and proinsulin.
Proinsulin consists of: an amino terminalB chain, a carboxy-terminal A chain and a connecting peptide called a Cpeptide. It is stabilized by three sulfide bonds. Inthe endoplasmic reticulum, proinsulin is folded and is transferred into theGolgi apparatus, which is then put into secretory vesicles. It is exposes the specificendopeptidases which cuts the C peptide, which creates a mature form of insulin.
Whenthe beta cell is stimulated, insulin is secreted from the cell by exocytosisand diffuses into islet capillary blood. In the endoplasmic reticulum, proinsulinis exposed to specific endopeptidases which cuts the C peptide, which generatesa mature form of insulin. Insulinand free C peptide are packaged in the Golgi into secretory granules whichaccumulate in the cytoplasm.Insulinsecretion is also secreted by amino acids such as leucine, arginine and lysine.Anfinsenexperiment couldn’t work on mature insulin because once insulin is fully synthesized,a precursor is cut down to the biological active state after forming.
So once the insulin is mature, it won’t foldthe same way into the same structure as it would before it matured, so theprotein isn’t active therefore you it cannot undergo Anfinsen’s experiment.