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Background

Insulin is the major anabolic hormone in all 'higher' organisms involved in regulating the uptake of glucose by cells of the body, amino acid synthesis and the conversion of carbohydrate into triacylglycerols.

In diabetes mellitus, due to failure of insulin secretion or action, patients are unable to utilise glucose properly and also to synthesise triacylglycerols from carbohydrates or amino acids. Symptoms include excessive thirst (caused by the changed osmotic potential of the blood), excretion of glucose in the urine (makes it taste sweet) and loss of weight (as body energy reserves are depleted). Demonstration of the role of the pancreas in diabetes mellitus was first provided by Mering and Minkowski in 1889 when they produced the condition in a dog by removal of the pancreas. However, attempts to extract the implicated hormone did not succeed until Banting and Best prepared an active extract from the Islets of Langerhans which they named insulin. The discovery of insulin dramatically changed the outlook for millions of diabetics worldwide, who previously would have died a few years after the onset of the illness but now could be stabilized on daily injections of insulin purified from pig or cow pancreas (porcine and bovine insulin respectively).

The Primary Structure of Insulin

The complete amino acid sequence of porcine insulin was determined in a herculean feat by Sanger and co-workers in 1955. Porcine insulin was shown to contain 51 amino acids, arranged in two chains (an acidic A-chain of 21 residues and a basic B-chain of 30 residues).

Here are the amino acid sequences of the A and B chains of porcine insulin:

• a) A6-A20, A7-A11, B7-B19
• b) A6-A7, A11-B7, A20-B19
• c) A6-A11, A7-B7, A20-B19
• d) A6-A11, A7-B7, A20-B7

The Secondary Structure of Insulin

Circular dichroism (CD) has been used widely in the study of insulin conformation in solution.

• A Short Primer on Circular Dichroism

Here is the far UV spectrum of insulin:

• a) alpha-helix
• b) beta-sheet
• c) random coil
• d) a mixture of all of the above

The Tertiary Structure of Insulin

• A Short Primer on X-ray Crystallography

Although insulin was first crystallized and made available for clinical use by Abel and co-workers in 1925, it was not until 1934 that Scott discovered that the rhombohedral crystals were a zinc-insulin complex. Together with Fischer, Scott showed that the zinc might be replaced by other divalent ions. In time, this opened up the possibility that the crystal structure might be solved by isomorphous replacement. An initial X-ray analysis of porcine zinc insulin at 2.8Å was completed in 1969 by Dorothy Hodgkin and co-workers in Oxford. (Click here for the obituary of Dorothy Hodgkin by Max Perutz, at the Crystallography section of the World-Wide-Web Virtual Library.)

The insulin structure was refined to 2.5Å by the Peking Insulin Structure Research Group in 1971. Much of the recent work on insulin structure has been carried out by Professor Guy Dodson and colleagues at York University in collaboration with Novo Nordisk of Denmark, by Professor Michael Weiss and colleagues in Chicago in collaboration with Eli Lilly and by Professor Axel Wollmer and colleagues in Germany.

2-zinc insulin has now been refined to 1.1Å resolution; in this structure, the positions of water molecules determined from neutron scattering are included.

The 2-Zinc Insulin Crystal (Porcine Insulin)

Click on one of the following to obtain the PDB structure file 4ins from the copy of the PDB nearest to you: [Birkbeck | Brookhaven | ExPASy]

In this crystal form, the unit cell contains 2 zinc ions and 6 insulin molecules. The asymmetric unit consists of 2 insulin molecules, (designated 'molecule 1' and 'molecule 2'), which are different. The asymmetric unit is presented and the hexamer can be generated by applying a three-fold symmetry operation through the crystallographic axis on which the two zinc ions lie.

Discussion Topic 1

Does this crystal structure contain

• (a) any water molecules?
• (b) any other solvent?
• (c) any counter-ions?
• (d) any other small molecules?

We are going to study the secondary structure of molecule 1. Download the following script file (4ins_sc1.txt), and input into RasMol, with the command:

script <path>4ins_sc1.txt

Discussion Topic 2

Use the RasMol command 'structure' to calculate the secondary structure according to the Kabsch and Sander's hydrogen bond-based DSSP algorithm. How does this compare with the secondary structure designation in the PDB file?

Many references use a colour scheme of blue for the A-chain and red for the B-chain. Use the following commands to achieve this (assuming you have already run 4ins_sc1.txt, and to remove the labels (you can copy and paste these into your RasMol command line window, or download and then input to RasMol the file 4ins_sc2.txt).

select *a
colour blue
label off
select *b
colour red
label off

The Disulphide Bonds of Insulin

select cys*a, cys*b
wireframe 80
ssbonds 80
select sulphur
colour yellow

• a) All the cysteines are connected in disulphide bridges, there is no free sulphydryl group so it is unlikely that insulin binds to its receptor by forming a disulphide bridge.

• b) Because of the proximity of A6 and A7 cys, reduction of the disulphides and refolding of insulin gives a (A6-B7,A7-A11,A20- B19) disulphide isomer by-product.

• c) Because of the proximity of A6 and A7 cys, reduction of the disulphides and refolding of insulin gives a (A6-A7,B7-A11,A20- B19) disulphide isomer by-product.

• d) The A7-B7 disulphide is partly exposed on the surface of the molecule

The Glycine Residues of Insulin

• a)A1

• b)B8

• c)B20

• d)B23

Discussion Topic 3

Why are the three glycine residues with positive phi dihedral angles important for the insulin fold?

The Core of the Insulin Molecule

A6 Cys, A11 Cys, A16 Leu, A20 Cys, B15 Leu, B19 Cys

• a) The side chains of these residues stabilize the insulin fold by being buried in the core of the molecule

• b) The side chains of these residues make the insulin fold unstable because they would like to hydrogen-bond with the solvent

• c) The backbone atoms of all these residues are buried in the core of the molecule

• d) All Cys and Leu side chains in the insulin molecule are buried in its core

The Surface of the Insulin Molecule

restrict *a,*b
wireframe off
ribbons off
spacefill
colour white
select sidechain and 24-26b
colour cyan
select sidechain and (13-14a,1-2b,14b,18b)
colour green

• a) The hydrophobic aggregation surfaces could explain why insulin is a "sticky" molecule which tends to adhere to glassware and to the insides of insulin infusion pumps.

• b) Because of their importance for the storage of insulin, these hydrophobic surfaces would be expected to be conserved in insulins from different species.

• c) The hydrophobic aggregation surfaces are on one face of the insulin monomer.

• d) The hydrophobic aggregation surfaces are on opposing faces of the insulin monomer.

How could insulin delivery by infusion pumps be improved?

The Insulin Dimer

select protein
wireframe off
spacefill off
label off
ribbons
select *a
colour blue
select *b
colour red
select *c
colour cyan
select *d
colour magenta

The orientation of B25 Phe

• a) The side chains of both residues are packed against the body of their respective monomers.

• b) The side chains of both residues point into the solvent, away from the body of their respective monomers.

• c) The side chain of molecule 1 turns away from the body of the molecule and contacts its partner in molecule two across the approximate 2-fold crystallographic axis.

• d) The side chain of molecule 2 turns away from the body of the molecule and contacts its partner in molecule one across the approximate 2-fold crystallographic axis.

Now look at the dimer in the direction perpendicular to the approximate two-fold axis (Figure 1).

Note that the residues involved in dimerization are in the beta- stranded (B24-B30) structure of the monomer.

• a) Residues B24 Phe and B26 Tyr of molecule 1 are hydrogen-bonded to residues D26 Tyr and D24 Phe of molecule 2.

• b) Hydrogen-bonding between the two insulin monomers results in the formation of a parallel beta-sheet structure.

• c) Hydrogen-bonding between the two insulin monomers results in the formation of an anti-parallel beta-sheet structure.

• d) The stability of the insulin dimer is further enhanced because it allows non-polar side chains in the monomers to become buried.

Introducing the Quaternary Structure of Porcine Insulin

In the presence of zinc ions, three identical insulin dimers assemble into a hexamer in which two zinc ions lie on the central three-fold axis. The approximate two-fold axis within each dimer is perpendicular to and intersects the three-fold axis.

The hexamer has been generated for you by applying a rotational symmetry operation about the three-fold axis to the dimer (the asymmetric unit); the solvent molecules are not included.

Obtain the hexamer structure file from either Birkbeck, which will be served with chemical MIME, or from the BNL or DL mirror (not chemical MIME-stamped).

The chains have been labelled as follows:

The following commands (script file 4ins_sc6.txt) display the 6 monomers as ribbons (A-chains in blue, B-chains in red). The 6 B10 His side chains are shown and the 2 zinc ions are coloured green.

select
wireframe off
ribbons
select *a, *c, *g, *i, *m, *o
colour blue
select *b, *d, *h, *j, *n, *p
colour red
select his10
wireframe 80
select zinc
spacefill
colour green

• a) Each zinc ion is co-ordinated by three B10 Histydyl nitrogens.

• b) The 2-zinc insulin hexamer is torus (doughnut) shaped

• c) The polar channel down the centre of the hexamer allows zinc ions to diffuse freely into and out of the assembled quaternary structure.

• d) The channel down the centre of the hexamer is penetrated by the polar side chains of B10 His, B9 Ser and B13 Glu.