bridges can be disrupted by treating a protein with 2-metaptoethanol. The bond
between two sulfurs can be broken and a new bond can be created between the two
sulfurs at the end of the two molecules of 2-merapthoethanol. The aim of
Anfinsen’s experiment was to show that the information for protein folding
resided 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 denaturant
urea and 2-metaptoethanol. Urea is a chaotropic agent which lets water molecules
to solvate non-polar groups inside the protein which disrupts the hydrophobic interactions.
protein then unfolded when this was added. It lost is original conformation and
become further inactive due to the enzyme’s active site changing. It refolds spontaneously and regains biological
activity when you remove the urea and 2ME from the solution.
the 2-metapoethanol but not urea led to recovery of 1% of the activity. This is
due 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 disulfide
isomerase (PDI)- an enzyme that catalyzes reduction of incorrect disulfide
bonds and allows a protein trapped in an incorrect conformation to unfold and
diagram above shows the overall steps of the Anfinsen experiment, it shows the
native ribonuclease A being added to urea and 2ME which is then denatured and
the disulfide bonds have been reduced.
is a 51 residue protein that is composed of two peptide chains linked by two
disulfide bonds. It plays a big role in the regulation of human metabolism. Insulin
is synthesized in rough endoplasmic reticulum in the beta cells which are
located in the pancreas. There are different types of insulin, there is rapid
acting, short acting and long acting. The structure of it determines how easy
it can fold, trafficking, self-assembly and receptor binding.
It is synthesized by precursor cells which
include preproinsulin and proinsulin. Proinsulin consists of: an amino terminal
B chain, a carboxy-terminal A chain and a connecting peptide called a C
peptide. It is stabilized by three sulfide bonds.
the endoplasmic reticulum, proinsulin is folded and is transferred into the
Golgi apparatus, which is then put into secretory vesicles. It is exposes the specific
endopeptidases which cuts the C peptide, which creates a mature form of insulin.
the beta cell is stimulated, insulin is secreted from the cell by exocytosis
and diffuses into islet capillary blood. In the endoplasmic reticulum, proinsulin
is exposed to specific endopeptidases which cuts the C peptide, which generates
a mature form of insulin.
and free C peptide are packaged in the Golgi into secretory granules which
accumulate in the cytoplasm.
secretion is also secreted by amino acids such as leucine, arginine and lysine.
experiment 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 fold
the same way into the same structure as it would before it matured, so the
protein isn’t active therefore you it cannot undergo Anfinsen’s experiment.