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


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Transient Linear Hydration Analysis

Ribonuclease A is a digestive enzyme which is released from the pancreas and cleaves ribonucleic acids at uridine and cytidine nucleotides.53 It is considered a “killer enzyme” because it functions so rapidly and efficiently. Since its surface is covered by cationic peptides, like lysine and arginine, it is extremely stable to acid and easy to isolate and purify.54 Literally thousands of studies have been performed on this small stable 124- peptide protein, including the classical folding study performed by Anfinsen in 1961.44

The ribonuclease A enzyme with the preferred cubic hydration patterning around the molecule.

As shown on the left above, four disulfide linkages tie coils and linear segments into an extremely stable structure.55 Before refolding experiments could be performed by Professor Anfinsen, the disulfides had to be reduced to sulfides by the addition of mercaptoethanol and urea had to be added to disrupt the ordering in surface hydration.56 As the compact protein unfolded and formed an extended filament of polypeptide, viscosity increased and enzymatic activity was lost. However, when the solution was dialyzed to remove the urea and mercaptoethanol and then exposed to oxygen in the air, the viscosity decreased and the protein regained virtually all of its catalytic activity. Of course, this meant that the polypeptide in the natural acidic medium had spontaneously folded into its native conformation with all of the sulfides in precisely the proper spatial positions to form correct disulfides, the same as in alpha-chymotrypsin described above.

However, when urea was left in the reassembly medium and the solution was exposed to air, only 1% of enzymatic activity was restored. Since the viscosity decreased, disulfides apparently reformed, but not in the native spatial positions. Only when surface water was permitted to form normal surface properties with proper patterning elements did polypeptide strands wrap spontaneous to form the single native structure. As illustrated on the right above, you can see that helical coil A is parallel with diagonal linear elements in the cubic lattice while B and C are parallel with those in horizontal planes.

Peptides 1-13

Transient elements of hydration on the front and right-side of the helical coil.

Selecting peptides 4 to 13 as the nucleating core, three of the peptides on the upper surface are in positions to hydrogen bond with water as a dielectric element as shown above.26 Peptides in coils often serve as binding sites in receptor proteins and enzymes. In this case, lysine 7 will serve as a cation with the counter-ion on histadine 12 serving as the anion. The amide group of glutamine 11, by hydrogen-bonding with water molecules at multiple angles (like the amides in urea) will destabilize the linear element and introduce an element of disorder and reversiblity to the binding site.

Peptides 1-25

Like insulin, the continuing segment of twelve peptides forms a loop composed of eight small hydration-disordering peptides with two alanines in the middle and a tyrosine on the end to attach coil A to coil B.

Polypeptide segment 15 to 25 contains multiple serines to provide flexability to the chain.

Although the hydroxyl groups of the serines are close to cubic patterning positions, they most likely disrupt surface hydration order and provide mobility to the chain. Since the chain is long, it not only permits coils A and B to form independently and avoid premature binding of hydrophobic surfaces but, as illustrated below, permits coil B to swing smoothly into position in the hydrophobic back side of coil A to form a firm union with arginine 33 coupled with glutamate 2 and all ordered water between the coils released.

Peptides 1-36

The 1-12 coil is directed into position by the ionic charges on the glutamate and arginine peptides.

Once coils A and B are in place, several serine hydroxyls on the right side are in positions to stabilize layering and cubic patterning in adjacent water.

The Binding Site and Ordering of Surface Hydration

As assembly continues, flexible segments fit beta sheets and coils together to release transiently-ordered water. The result is an extremely stable protein with a planar ionic space between elevated sides. Just as ionic head groups on coil A supported a bridging element of dielectric linear hydration, additional ionic head groups support a dynamic hexagonal patterning of water molecules on the surface of the central plane.

Water molecules bridging between the surface groups hold them in proper positions for RNA binding.

Seven amines, three acids and four polar head groups of peptides provide the binding site for the attachment of ribonucleic acids and the cleavage of their bonds.  The circled area on the right above is a depression in the surface of the binding site which is just large enough to accommodate three water molecules. It is the pocket for selectively binding the basic rings of uridine and cytidine nucleotides.

Ribonucleic Acid Binding

RNA binding takes place on the flat planar upper surface of the enzyme.

On the left above, the phosphates of five nucleotides are illustrated bonded to cationic amines at peptide positions 7, 39, 41, 66, 98 and 119.  As illustrated below, the central uridine nucleotide is hydrogen-bonded between histadine 119 and lysine 41 while the 2’ hydroxyl oxygen atom on the ribose ring of that nucleotide is hydrogen bonded to the basic ring of histadine 12 below. The basic ring of that nucleotide is hydrogen-bonded to the hydroxyl group of threonine 45 in the depressed pocket circled on the right above.57

Nucleotide Binding

A uridine nucleotide binds into the reaction site with its phosphate bound between lysine 41 and histadine 119.

This enlargement of the uridine nucleotide in the reaction site, illustrates how the cationic ring of histadine 119 (at A) has moved over one water unit from its position in crystalline protein to hydrogen bond with the 5’ oxygen of the next nucleotide. As mentioned above, the basic ring of histadine 12 is hydrogen-bonded to the 2’ oxygen of the uridine nucleotide below the ribose ring at B.  The cationic nitrogen of lysine 41 is hydrogen-bonded to the anionic phosphate oxygen of the uridine nucleotide.

Catalytic Cleavage

Top Views of the three-stage catalytic cleavage reaction.

With two oxygens of the central phosphate protonated by cationic amines at 41 and 119, the bond between the 5’ oxygen of the next nucleotide and the phosphorus atom brakes and forms a cyclic phosphate with the 2’ oxygen of the ribose ring. However, as the 5’ hydroxyl group leaves, a water molecule moves in between it and the amine on lysine 41, as shown in Figure 2, and reacts with the cyclic phosphate to form the free acid as shown in Figure 3.

It also can be seen in Figure 3 that the binding site is so perfectly “designed” that the next nucleotide in the chain is oriented perfectly to move into the reaction site for cleavage. The site operates like a zipper, feeding pyrimidine nucleotides, one after another into the reaction site, skipping over adenosine and guanosine nucleotides which do not fit and moving on to the next pyrimidine. With its broad planar surface and perfectly-positioned cationic peptides, ribonuclease A is truly a “Killer Enzyme.

In spite of the fact that the principles of surface hydration presented here differ substantially from present concepts, they provide rational explanations for many of the unanswered questions in molecular biology today. Hopefully, this presentation will encourage others to take more seriously the possibility that it may be the dynamic linearizing properties of surface water which assist in the spontaneous folding and assembly of natural sequences of polypeptides to form functional proteins.

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