Protein Structure
Carboxypeptidase A
Alpha Chymotrypsin
Ribonuclease A
Receptor Sites
Double-Helix B-DNA


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Transient Linear Hydration Analysis

One of the first proteins to be analyzed in detail by X-ray crystallography was insulin.34 As mentioned previously, it is a small protein which is formed as a single linear strand of polypeptide within the beta cells of the pancreas and folds spontaneously into three distinct regions.

An alpha-carbon view of insulin molecule assembly.

Region A is composed of two coils tied together by a short linear segment. B is composed of a central long coil with two linear segments at each end. C is a 31-peptide linear unit containing multiple small peptides which are highly hydrated to permit mobility. It permits A and B to fit tightly together, release ordered surface water and form the anhydrous central core of the  molecule. The terminal unit, D, ties the polypeptide to a transport protein which transports it through the outer membrane of the synthesizing cell. Segments D and C are removed enzymatically before the insulin molecule is released from the beta cells into the blood stream. Insulin’s structure is such that six units combine to form a hexamer in its dried crystalline form and it circulates in the blood as both a monomer and a dimer. The crystalline form, which was used for X-ray analysis, was stabilized by a zinc ion in the middle of the hexameric complex.34

Top and Front rotational views of the insulin molecule in the Quantized Cubic Hydration Matrix.

As mentioned before, the insulin molecule, in contrast to most water-soluble proteins, reflects cubic patterning and geometry in its external structure. In order to better illustrate that, the molecule is rotated 60 degrees in the standard cubic hydration lattice. Clearly, in all views, the longest coil parallels diagonal transient linear elements of surface water. Of course, the reason for this external cubic patterning is that it is a regulator hormone which binds to a cubically hydrated membranal receptor protein. By displacing transient linear elements of hydration and binding tightly to multiple sites, it initiates the transport of glucose into cells and a number of other essential physiological functions.35

B-Chain Peptides 1-21

As can be seen in the upper illustrations, the 10-unit polypeptide in unit B was selected as the nucleating core for assembly. It contains a series of hydrophobic peptides, wraps rapidly into a coil and, like alpha chymotrypsin, produces a dipolar unit with histadine 10 at the top and glutamate 21 below. Serine at the top and glycine below break the coil with the carbonyl oxygen of glycine 20 bridged to the phenolic oxygen of tyrosine 16 by a single water molecule in plane -4. Peptides in front and back of the coil are hydrophobic and linearize water because they do not hydrogen-bond with it, while those on the right in the Front View linearize water as dielectric elements to delocalize charges between the ionic peptides.

Transient linear elements of hydration form on the hydrophobic faces of the B unit of the insulin molecule.

Once the coil forms and cubic patterning is established, the initial segment of the B chain, which remains as a linear segment because it contains two adjacent amide peptides, follows the orientation of linearizing water positioning the methyl groups of leucine 6 above the coils with segment between phenylalanine 1 and asparagine 3 bent down to position the aromatic ring adjacent to valine 18. The polar amides of asparagine 3 and glutamine 4 are positioned above and below the chain in Planes 2 and 0 to disrupt transient linear hydration adjacent to the chain and provide a degree of freedom and an increase in solubility. Notice in the Top View that most of the lipid peptides are still clustered on the upper and back sides of the coil to provide a space for the complimentary region of segment A to displace transiently ordered water.

B Chain Peptides 1-30

The continuing B chain, from glutamate 21 to alanine 30, fits into the hydrophobic space behind the coil. To more clearly show its structure, it is illustrated as an independent unit in the upper views and then behind the coil in the lower views.  Phenylalanine 24 fits into a hydrophobic space behind the coil, while phenylalanine 25 fits below the methyl group of threonine 27.

The linear 21 to 30 segment of polypeptide fits into the hydrophobic back side of the coil displacing orderd surface water.

Note in the upper right Front View that the cationic amines 22 and 29 and anionic acids at 21 and 30 extend out away from each other rather bending toward each other. Peptides with highly-charged groups often are far apart in protein structures because water is so effective at delocalizing charge. Water, by clustering around charge centers, disrupts local transient linearization and permits the groups a good deal of freedom.

As mentioned before, the alignment of a linear segment next to a coil is an extremely common structural feature in proteins. However, in this case, the aromatic ring of phenylalanine 24 forces the continuing segment to angle away from the coil as shown in the Top View above. By fitting the aromatic ring at 24 next to the coil and the oxygen of threonine 27 into the cubic lattice, the phenolic oxygen of tyrosine 26 is in a position to bridge by a water molecule to the oxygen of glycine 8 (as seen in the Top View on the lower left). In the same Top View it can be seen that the entire center of the molecule is hydrophobic – too unstable to remain open.

The A-Chain

The A unit of polypeptide is unique in that it is composed of two short coils which are tied together by a short linear element of four peptides beginning and ending with serines. Cysteine 6 bonding to cysteine 11 ties the two coils tightly together with a linear hydrophobic face on the lower right as illustrate in the Front View.

Two short coils are attached together by a short linear segment to form the A unit.

The C-Chain

The C polypeptide is a tether between A and B. With thirteen glycines and a proline, it is highly hydrated and extremely flexibility. In fact, the three glycines at positions 28, 29 and 30 provide sufficient flexibility to permit the hydrophobic side chains of leucine 24 and 26 to displace ordered water on the right side of the A unit and provide stability for transport to the B unit.

The C-chain tether brings the A and B units together.

Another feature of the tether is that it is highly acidic with several pairs of acidic peptides which are close enough together to share a negative charge. Sodium or calcium ions must surround the segment to neutralize the negative charges. Notice that cubic patterning is included only around the A and B units to illustrate that random water most likely is around the tether. Although the C chain must be extremely flexible, it most likely forms beta-sheet hydrogen-bonding between the segments to provide transport order.

At this stage in the description of insulin assembly, it is important to realize that the interpretation which has been provided, particularly the role of the C unit, has not been described before and has been made possible only by including water and its property of forming transient elements of order and cubic patterning around assembling units.

The TLH Insulin Model

After cleavage of the C unit, the only hydrophobic region on the surface of the molecule is the diagonal space between terminal alanine 30 and the B coil. Although water most likely forms dielectric linear elements along the lower right-hand side of the molecule in the Front View, the upper left side is covered with amide and amine groups to disrupt linear order and increase solubility.

A transient linear element of water forms across one face of the insulin molecule.

Insulin Receptor Binding

By rotating the molecule 30 degrees to the right, as shown in the Side View on the right below, a planar hydrophobic face of the molecule is revealed with layering of water adjacent to the aromatic rings of phenylalanines 24 and 25 and the methyls of valine 12.   

A flat planar hydration-ordering face of the insulin molecule binds to an hydration-ordering site in a receptor protein.

It is this hydration-ordering face of the molecule which a recent study has revealed binds strongly to receptor protein in membrane to activate glucose uptake into cells.35 The lysine/alanine carboxylate end of the B chain (at 29 and 30) extends into the receptor protein with the aromatic rings at 24 and 25 and methyl groups of valine 12 making contact with hydrophobic peptides in the receptor site.

Viewing that broad flat hydration-ordering B-face of the insulin molecule above, it is not hard to imagine that the binding surface of the receptor binding site is covered by covalent transient linea elements of hydration when not occupied by the insulin molecule. Once again, it appears that transient linear hydration and cubic hydration patterning may play a role, not only in the assembly of the protein but in its functional properties as well.

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