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


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Transient Linear Hydration Analysis

Chymotrypsin is synthesized on the ribosome as a single polypeptide strand of 245 aminoacid residues which fold spontaneously into the finished protein called chymotrypsinogen.43 However, this form of the enzyme is not active. Enzymatic cleavage of peptides 15 and 16, followed by removal of several other peptides in the chain, yields the finished active stable enzyme, alpha-chymotrypsin. Since the body of the enzyme is not affected by the activation stages and our analysis of the folding begins with peptide 245 at the acid end, this presentation is valid for all three forms of the enzyme. Only the final, active form will be shown.

The terminal coil of the polypeptide of the alpha-chymotrypsin enzyme was used as the nucleating core.

Once again, this analysis will begin with the longest coil but, this time, its upper face is hydrophilic. Although the coil is not parallel with any of the crystallographic axes, it is parallel with transient linear elements in the quantized cubic matrix. Thus, coordinates reported for the crystalline form were used directly for comparative anaysis.43 Although analyses of both enzymes in this article could be performed without rotating them within their crystallographic axes, some which have been analyzed did require rotation to provide for best fit of coils or beta sheets within the cubic lattice.

Peptides 245 to 230

Like carboxypeptidase A, alpha-chymotrypsin cleaves polypeptides – but not from the end - instead, it hydrolyzes them in the middle at aromatic peptides. The reaction site in the enzyme and the mechanism of cleavage are different from that of carboxypeptidase A but the principles of hydration are the same.

Cubic hydration patterning around the alpha-chymotrypsin enzyme is defined by the terminal coil.

Sometimes, polypeptides which emerge from ribosomes remain as linear segments but those on the acid end of chymotrypsin are so hydration-ordering that they, like those of carboxypeptidase A, wrap rapidly into a coil. However, in this case, the coil is broken by arginine 230 which is highly charged and highly hydrated. The coil is a polarized dipole with terminal anionic asparagine 245 at one end and arginine at the other. The opposite charges tend to be neutralized by quantized coupling fields which most likely radiate around the coil. Neutral glutamines 239 and 240 increase hydration disorder and entropy on the back and upper sides of the coil while the hydrophobic methyls of valines and leucines on the lower front produce ordered voids to direct the continuing polypeptide chain away from the dynamics of hydration toward the voids.

Whenever there is a cluster of water-ordering groups on one side of a coil, like the methyls and aromatic ring on the front side of the one shown above, there is the possibility that a linear segment of polypeptide further down on the chain will align next to it with its hydration-ordering groups next to the coil. If you recall, the initial structural complex in carboxypeptidase A had a coil/linear-segment structure. However, if the continuing chain does not contain a linear segment with the proper side groups, it will follow an ordering element of water molecules in cubic patterning (usually at 60o) away from the coil. As will be seen below, the continuing chain of alpha-chymotrypsin contains an order-breaking proline and a series of small hydrating peptides; it would be expected to move away from the coil rather than aligning next to it.

Peptides 245 to 215

In this Top View, you can see that the chain does move away from the highly-charged arginine 230 to tyrosine 228 which hydrogen bonds its phenolic hydroxyl into transiently-ordering plane -2. As noted before, phenolic hydroxyl groups of tyrosine peptides, with their relatively high degree of rotational freedom, play extremely important roles, both in the assembly of proteins, as well as their functions. Tyrosine 228, by stabilizing transient hydration in plane -2, provides stability for a loop of small polar peptides to permit the indole ring of tryptophane 215 to rest on the methyls of valine 227.

The continuing chain follows cubic patterning out at a 60 degree angle to form a linear-water disrupting loop.

The Front View illustrates how the chain winds down in a wide loop following valine 227 using multiple threonines and serines to provide randomness in water to turn the chain around and permit the sulfhydryl of cysteine 220 to hydrogen bond into tyrosine-stabilized plane -2.

Since we have begun the analysis of a new protein, it is important to be reminded of the experimental evidence for the formation of transient two- and three-dimensional cubic order adjacent to non-hydrogen-bonding surfaces. Remember, pure water can be cooled to as much as 30o below zero centigrade without crystallizing. However, if the water is adjacent to a lipid surface or one with properly positioned hydroxyl groups, supercooling is impossible – crystallization occurs immediately at 0oC.22 Hexagonal surface patterning induces transient two- and three-dimensional hydration ordering and that, in turn, induces quantized cubic patterning order around the molecule. Although only short linear hydrogen-bonded elements are present at any instant and water molecules occupy only probability positions, they guide the folding of polypeptides.

Peptides 245 to 202

With the methyls of isoleucine 212 positioned below tryptophane 237, the chain drops down at glycine 211, goes under the coil and forms a long loop below the coil with the nitrogen of tryptophane 207 hydrogen-bonded into plane -4 and the cationic amine of lysine 202 into plane -5. Again, we must be reminded – even though linear elements of water are displayed as extended linear elements and layers, only short units of order are present at any instant. Only when viewed over time, would the probability positions of quantized hydration order be evident.

The polypeptide chain forms a second loop adjacent to the first below the initiating coil.

With that view in mind, transient linear elements of hydration might well form continuously adjacent to the hydrophobic surfaces between charged asparagine 245 and lysine 202.

However, it is unlikely that the hydrophobic cavity shown in the Front View would be stable – too much water would be trapped in the space. If that area is not filled rapidly by a complimentary hydrophobic segment of polypeptide, crystalline water might form in the cavity. Thus, it is likely that the 202-211 loop would initially form behind the front loop, more in line with the coil, and then move into the position shown above as lipid groups move into the cavity. To provide accurate estimates of the effects of surface hydration on stability, detailed calculations of the thermodynamics of each hydration state will be required. Until such methods are applied, we will be forced to rely on more-simplistic, general interpretations.

Peptides 245 to 182

Once the chain has turned at lysine 202 and passed back under the coil, it travels upward at proline 198 and through two glycines to serine 195 which is tied tightly in plane 0 by a disulfide bond between cysteines 191 and 220. To complete this section, the chain turns 120o at cysteine 191 and passes below mostly water-ordering peptides to form a water bridge between anionic cysteine 182 and cationic arginine 230.

After passing under the coil, a water-retaining cavity is left in the front region of the rectagonal assembly.

In the Front View, it can be seen that the polypeptide chain, by wrapping back and forth, has essentially filled an open space displacing multiple linear elements of transiently ordered water molecules with serine 195 in the same plane as serine 218. It is as if serine 195 has been placed on the corner of a block of atoms, in precisely the proper position, to perform an important function. And, indeed, it will! As assembly continues on the right side, peptides will replace the linear element of water leading into serine 195 and provide binding for substrate polypeptides. Serine 195 will be held by the block in precisely the proper position to serve a primary role in catalytic hydrolysis. The region designated as “A” is an open space, with two serines and a threonine on the left side, where water will be displaced by the aromatic rings of substrate polypeptides.

Peptides 245 to 133

Since proteins have complex structures and, sometimes, do not have coils to serve as reference units, alpha-carbon plots may be used instead. Viewing the plot to peptide 133, it can be seen that the chains, in wrapping back and forth, follow cubic hydration patterning.

The alpha-carbon plot of alpha-chymotrypsin protein illustrates how it conforms with cubic patterning.

In the Front View of the total protein on the right, the molecule is divided into six spatial units, each with cubic patterning. It would be interesting to see if natural proteins are composed of a finite number of hydration-stabilized cubic space units which combine in different combinations and in different orientations to form finished functional proteins.

As illustrated in the Front View on the left, individual chains do not necessarily follow the orientations of linearizing surface water but the assemblies which develop in the internal regions of the proteins do reflect cubic patterning. Whenever linear chains change direction, there is usually a glycine, serine, proline or asparagine at the position of change to disrupt transient linear hydration order, increase hydration entropy and drive the chain in an alternate direction.

The Transient Linear Hydration Model

In these views of the total protein, surface groups are included to illustrate that most of them are in random positions relative to cubic probability. As mentioned many times before, the primary purpose of these groups is to disrupt transient linear hydration in surface water and increase solubility.

The alpha-carbon plot of alpha-chymotrypsin protein illustrates how it conforms with cubic patterning.

Although surface groups and chains in the crystalline form, as viewed above, are held in relatively ridged positions, they have a good deal of freedom in their natural state as water molecules bridge between them in dynamic fashion. For example, the upper loop in the Front View has a degree of flexibility but the one bearing histadine 57 is tied by the adjacent disulfide link to the larger unit and is relatively ridged. The diazo-ring of histadine 57 can rotate up and down as it participates in the hydrolysis reaction, but the peptide itself has relatively little freedom to move.

Since this molecule contains only two stabilizing coils, it is the disulfide linkages, which are formed by oxidation of cysteine sulfides during assembly, that provide much of the internal stability. As mentioned in the introduction, it is essential to realize that, in the folding of the alpha-chymotyrypsin polypeptide, 10 sulfides must be held in precisely the proper spatial positions to form the correct five disulfides. Unless some sort of surface hydration patterning is involved to hold them in proper positions, it is difficult to understand how proper assembly would be possible. If the ordering property of surface water were disrupted by the addition of an amide like urea to the aqueous medium, as it was in the classical Anfinsen study,44 one would expect random disulfide formation and a molecular mess to be produced. Some sort of order in surface water must be involved in directing the folding and assembly of this enzyme!

The Catalytic Reaction Site

As illustrated below, the primary role of alpha-chymotrypsin in digestion is to hydrolyze polypeptides at aromatic peptides like tyrosine and phenylalanine. The space designated as “A,” which was left open and hydrated during the folding process, is precisely the proper size to fit the aromatic rings of bound peptides as they are held for hydrolytic cleavage.

The a substrate polypeptide is illustrated entering and leaving the catalytic reaction site of alpha-chymotrypsin.

In the Front View on the lower right below, it can be seen that the reaction site between peptides 57 and 195 is surrounded by peptides with aromatic rings at 215, 41, 39, 141 and 146 to increase the attraction and binding of substrate polypeptides with aromatic rings. Methyl groups on the corners of the site provide even greater order of water around the reaction site. As mentioned above, the aromatic rings of substrate polypeptides bind in region A while the substrate chain binds at B, C and D.

Top and Front Views of the reaction binding site of alpha-chymotrypsin.

Reaction Site Hydration

The X-ray diffraction pattern of this crystalline enzyme reveals a number of water molecules in binding site A but their high energy makes it difficult to define precise positions.43 When the protein is in its natural aqueous medium, bridging water molecules must be even more dynamic. However, water molecules in the binding site must have preferred probability locations to guide substrate molecules into proper binding positions. In order to locate those positions, physical and computerized models of the site were constructed with water molecules at acceptable angles and distances for covalent hydrogen bonding. The distribution shown below is one of several alternatives.

Hydration bridging is shown between polar and ionic peptides which form the reaction binding site

Water in front of the aromatic rings of phenylalanines 41 and 39 would be expected to be highly ordered and hydrogen bonded to the carbonyl oxygens in the polypeptide chain. Some water molecules are in cubic patterning positions, some are not but the positioning of aromatic rings at 39 and 41 above and below planes 0 and 1 permits the aromatic rings of substrates to complex with them as they move from one binding position to the next into the reaction site. Within cavity A, water molecules simply bridge from the hydroxyl of serine 195 to oxygens on the other side in a number of different bonding relationships before being displaced by an aromatic ring.

In the Front View, note that there is a continuous hydrogen-bonding link from aspartate 102, through the histadine ring and peptide 192, to the carboxyl of aspartate 194. This proton- and electron-coupled dielectric element plays a critical role in the hydrolysis reaction.43 The sulfur-containing chain of methionine 192 has been removed in the Front View to permit this element to be viewed. However, as shown below, it most likely plays an important role in directing aromatic rings into the binding site.

Substrate Binding

In the Top View on the left, the substrate is shown approaching the binding site. As pointed out above, it is highly probable that the aromatic rings of approaching polypeptides are attracted to the reaction site by the aromatic groups around it. Since aromatic rings readily form charge-transfer complexes with each other and with sulfides, it is likely that the aromatic ring of an entering substrate forms a complex with the sulfur atom of methionine 192, as shown on the right below, as it is escorted into cavity A.

Substrate polypeptide is drawn into tight dehydrated bonding in the reaction site.

Although substrate chains most likely approach the reaction site in many different orientations, they must initially form water bridges similar to those illustrated on the upper right and then move into firm binding as shown below. Bound, as shown, almost all transiently-ordered surface water in the site has been displaced. The aromatic ring is in the groove at A, the substrate chain is hydrogen-bonded to the biaryl chain on the right side of B, while, on the left, the chain is over the methionine 192 chain with one of the peptide oxygens bonded to the phenolic oxygen of tyrosine 146. The carbonyl of the peptide to be cleaved is held next to the oxygen of serine 195 at B at a 90o bend in the substrate chain with its nitrogen hydrogen-bonded to histadine 57.

If you recall, the plane of water which was displaced by the substrate polypeptide in the carboxypeptidase site was parallel to four serine hydroxyls. In this case, the displaced transiently-ordered water is adjacent to the 39-41 chain of aromatic rings 39 and 41.

Substrate Cleavage

As the tyrosine peptide in the substrate approaches the serine 195/histadine 57 complex, the proton on the oxygen of serine moves, in quantized fashion, to the nitrogen of histadine and the oxygen of serine bonds to the carbonyl carbon of the substrate peptide to form the tetrahedral intermediate, TI. As the C-N bond in the intermediate breaks, the right-hand portion of the substrate chain is released as a free cationic amine and the left side is left tied to the serine oxygen as an ester, E.43

Calalytic cleavage of the polypeptide substrate in the reaction site involves two steps.

Peptide 192, which is shown below the ester in the Hydration Figure on the lower left, holds the ester oxygen in precisely the proper position below water molecule w, that a second tetrahedral intermediate forms with the water molecule bound to the carbonyl carbon. When the bond between it and the serine oxygen breaks, the left-hand substrate chain, with its terminal peptide tyrosine, is released.


In the Front View shown below, it is clear that water molecule w, which is hydrogen- bonded to the nitrogen of histadine 57, is close enough to the carbonyl carbon of the ester to bond with it and perform the hydrolysis. It is also clear that the negative charge imparted to water molecule w from aspartate 102 through histadine 57 and the positive charge from peptide 192 facilitate the reaction.

In the second hydrolytic stage, a water molecule is held in precisely the proper position by histadine 57.

What is not displayed in the figures is that, in forming the initial binding complex, stress is imposed on the bonds which is released in forming the ester and conversion to the acid.43 However, it must be remembered that a driving force in the formation of the reaction complex, as well as the assembly of the protein, was the release of ordered water and increase in local hydration entropy.

Based on a concept of totally random disordered hydration around natural molecules, the probability that orderly functional systems could have evolved spontaneously on the early earth is zero.1 However, natural molecular assembly did not occur in a vacuum, it occurred in an environment which provided both randomness and order: randomness to provide for motion and change, order to provide for efficiency and reproducibility.

Thanks to Professors Lipscomb and Blow and coworkers for providing the structures and mechanisms of cleavage for the two enzymes presented above.42, 43, 51

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