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


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In order to perform a comparative analysis of the crystalline structure of a protein in the cubic matrix, it is necessary to select a segment of the protein as a nucleating core.47 As mentioned above, the primary function of external groups on water-soluble proteins, such as carboxypeptidase A and alpha-chymotrypsin, is to disrupt order in surface water. For that reason, there often is little resemblance between the external shape of the protein and the cubic lattice. Based on analyses of a number of proteins, the best nucleating unit is an internal coil. Coils form rapidly and usually are composed of peptides with water-ordering methylene alpha carbons on both sides.48 The carboxypeptidase molecule has been positioned so that the terminal coil ending in peptide 307 is parallel with transient linear elements of hydration in one plane of the cubic lattice as the Top View and perpendicular to that plane as the Front View.

The long coil at the carboxyl-end of the Carboxypeptidase A Enzyme was chosen as the nucleating core for analysis.

The long terminal coil in the crystallographic structure42 was used as the nucleating core and, as illustrated below, positioned for best fit of ordering groups on the coil in the cubic matrix realizing that, once preferred probability cubic patterning is established around the core, it is maintained as a directing influence throughout assembly and function of the enzyme.

Since hexagonal planes in the cubic lattice are not above one another, it is necessary to designate in the left-hand corner of the Top View which horizontal plane is being illustrated in the Front View. Plane 1 in the Front View on the right is displayed in the Top View on the left. Since every third horizontal plane is the same, water molecule positions in planes -3 and 3 are the same as those in plane 0. It is important to realize that hydration planes are displayed behind the molecule simply to provide a reference of probability positions of hydration patterning involved in directing assembly. A dielectric linear element of hydration is illustrated emanating from the enzymatic binding site in in plane 1 in the Top and Front Views above. Details regarding the binding and the reaction will be presented later. However, most surfaces of the carboxypeptidase protein, except for the linear lower left side, which binds with another molecule to form a dimer in solution, are covered with order-disrupting groups to increase solubility.

As folding and assembly proceeds, chains move through multiple low internal-energy states, with the continued loss of ordering surface water until the final protein is produced with essentially all internal water displaced and the polypeptide in low-energy state.

Peptides 307 to 283

As the carboxypeptidase-A polypeptide emerges from the ribosome, the 307 end rapidly forms the 307-283 coil unit to reduce the order in surface hydration. (See www.proteinhydration.com for detail.) In the list of peptides below, it can be seen that all peptides in the coil, other than glycine 295, are coil and sheet formers46 and that glycine has ordering peptides on both sides. Once helical coil formation begins, it continues until it reaches proline 288 which turns the chain and it continues in an extended coil to proline 282.

Hydration bridging between threonines 303 and 293 defines preferred cubic patterning for Carboxypeptidase A.

Covalent linear elements of hydration, which form repetitively on the sides, are illustrated as continuous elements realizing that only short segments of five to six water molecules are present at any instant, that they form in multiple cubic-patterned layers and last for only about 10-12 seconds. Like electrons around atoms, the water molecules are pictured in quantized probability locations. Note that it is the hydroxyl groups of threonine 304, threonine 293 and serine 284 which define the orientation of cubic patterning around the coil. The light blue and white carboxyls of amides 306 and 291 disrupt cubic patterning by hydrogen-bonding in multiple locations while carboxylates 307 and 302 disrupt cubic patterning by clustering water around them and directing covalent dielectric elements of hydration out from them to delocalize their charges. Although proline 288 terminates the tight coil, it continues in a slightly more open form positioning the methyls of isoleucine 286 and alanine 283 adjacent to the stabilizing linear elements.

The Front View illustrates how the anionic oxygens of glutamate 292 and the cationic nitrogens of histadine 303 above the coil are most likely stabilized by resonant proton “tunneling” through transient linear elements above the coil.32 Amides on the left and upper sides of the coil, by disrupting order in surface water not only increase solubility but, by elevating kinetic energy and thermal motion on those sides, spontaneously drive the coil in the direction of hydration ordering on the right side.

Peptides 307 to 263

As expected, the chain moves to the right and, at glycine 278, moves diagonally upward in a beta-turn to permit arginine 276 to couple by a water bridge to tyrosine 277 and reinforce the upward movement of the chain. Glycine 275 then introduces a second beta turn to permit arginine 272 to couple with glutamate 292 on the upper surface of the coil   and position the ordering side of the segment from arginine 272 to tyrosine 265 to displace ordered hydration on the right side of the coil.

Linear polypeptide segment 272 to 264 displaces the linear elements of water adjacent to the terminal 307 coil.

This permits the methyl groups of leucine 271 and the aromatic rings of two phenylalanines to fit perfectly into the hydrophobic space next to the coil with tyrosine 265 coupled with histadine 303 above the coil. Note that the side chains on the linear segment of polypeptide alternate from side to side permitting different hydration properties on each side. In this case, the left side is hydrophobic while the hydroxyl group of serine 266 on the right side is in precisely the proper position to support ordered cubic hydration in plane -1 with the hydroxyl of threonine 268 supporting linear order in plane -2. Cubic hydration patterning in plane 0 is reinforced by phenylalanine 279 and leucine 280 but interrupted by glutamate 270.

In contrast to the ends of the assembly where ion pairs turn the segments around, glutamate 270, by clustering water around it, disrupts the formation of linear elements parallel with the right side of the complex. As we shall see, glutamate 270 will force the continuing polypeptide chain to remain at the upper end next to the 268 - 264 segment.

Peptides 307 to 251

After placing the lipid group of isoleucine 263 in the ordered void below the assembly, as shown in the Front View, glycine 262 permits the chain to change direction and, with three order-forming groups, initiate a short new coil parallel to the initial one. This positions the carboxylate group of aspartate 256 in the same plane as that of glutamate 270 with covalent linear elements of hydration stabilizing a negative charge by proton tunneling between them. As the chain continues, serines play a major role. Serine 258 hydrogen-bonds to serine 266 to position the coil while the sequence, serine-glycine-glycine-serine, permits the chain to move upward and place the hydroxyl of serine 251 into the ordering elements in plane 0.

Polypeptide segment 262 to 258 forms a short coil with the hydroxyl of serine 251 in a cubic patterning position.

The Front View illustrates a transient linear ordering element of water on the right in plane 0. Once protein assembly is complete, this element will be replaced by side chains of peptides which will hold substrate molecules in precisely the proper position for hydrolysis. It is important to realize: once quantized cubic hydration patterning is established by elements of transient linear hydration next to the initial coil, a type of “Quantized Cubic Hydration Patterning” is established around the molecule to direct both assembly and function.20 Again, in the Front View, you can see that the ionic groups on the upper left are in a rather circular form. Further out in the polypeptide, a coil has already been generated with ionic groups which are complimentary to those shown above. In proteins containing a number of coils, most of them form early in the assembly process and fit together later to form the final protein.48 Again, it is important to point out at this stage of folding analysis that prolines, glycines and serines are positioned in precisely the proper positions in sequences to permit chain mobility and oppositely-charged ions and hydrophobic surfaces to couple together, release ordering water and form thermodynamically-stable units. During molecular selection in the earliest phases of random molecule formation, sequences of peptides which had proper turn points to permit the formation of thermodynamically-stable assemblies survived, those which could not assemble spontaneously were chewed up to form those that could. Thus, surface water not only directs folding and assembly today, it was intimately involved in the selection of the molecules which could function cooperatively in living cells.  

Water-insoluble membranal proteins are unique in that they often are composed of long coils which assemble in an aqueous medium with polar groups on the outside and non-polar, lipid groups inside. In contact with membranal phospholipids, the coils rotate so that the polar groups are on the inside forming a central pore through the membrane with non-polar groups on the outside in contact with the lipids. Peptides around central pores provide selective binding sites for water, molecules and ions to be transported in and out of cells.49 In fact, in a recent report, an element of transient linear hydration permitted the selective conduction of protons through a pore in a membranal protein.50

Peptides 307 to 241

In order to maintain stabilization of the negative charge between carboxylates 256 and 270, the continuing polypeptide chain loops over the transient linear element of water between them with the methyl groups of isoleucine 247 above it. The aromatic ring of phenylalanine 279, which also is in plane 0, increases hydration order next to the dielectric elements which radiate from glutamate 270.

Glutamate 270, by hydrogen-bonding strongly with water, forces segment 250 to 241 to form a loop.

In the Front View, it can be seen that the hydroxyls of tyrosine 248 and threonine 246, with only a slight upward movement of the chain, are in positions to reinforce cubic hydration patterning in plane 3 and provide more hydration space below the loop. Note how the polypeptide chain 251 to 248 tends to follow the diagonal plane of cubic patterning. With serine 251 positioned in the same horizontal plane as the two carboxyls, transient linearization is supported in plane 0 by all three peptides.

Even at this early stage of polypeptide folding, it is easy to see how the hydrated cavity below the methyls of isoleucine 247 might serve as a hydrophobic pocket for the aromatic rings of polypeptides which will bind for cleavage. Since the loop containing the cluster of methyl groups is free to rotate upward, it can open and close to admit either water or the aromatic rings of substrates. Of course, this is only a small portion of the catalytic site which will bind substrate polypeptides. Tyrosine 248, with its phenolic ring rotated downward, will be involved in hydrolytically removing the terminal peptides of substrates.51 Although a few of the peptides which will be involved in the hydrolysis are already present, many more will be needed to provide for firm binding and transport of substrate polypeptides in and out of the reaction site.

The Transient Linear Hydration Model

In the Top View of the finished protein, it can be seen that no less than three coils overlay the terminal one with a curved plane of linear beta-sheet segments wrapping around the right side. Although the choice of the terminal coil as reference proved satisfactory for the analysis, the other coils follow cubic patterning and could have been used instead.

Dielectric linear elements of water molecules emanate from the reaction binding site of the Carboxypeptidase enzyme.

In the Front View, it can be seen that, even though the coil/linear segment assembly at the upper left is not parallel with the horizontal cubic planes, the ionic ends of the peptides in it reach high enough to hydrogen-bond into plane 3. In fact, by moving into plane 3, space below the loop opens further for hydration. Also, it must be realized that this protein is not being produced in isolation - thousands of molecules, with the same structure, are being produced by the ribosome – one after another. Molecules must have external structures which permit tight binding for efficient storage but with sufficient hydration space between them for rapid release. The line of polar groups on the left side in the Top View permits diagonal hydration and the binding of two molecules to form a dimer in solution.

However, the most important area of hydration order, relative to function, is that illustrated on the upper right side in the Front View by water in plane 1. Until now, all atoms in the protein have been displayed in their X-ray crystallographic solid-state positions. However, in aqueous solution, polar groups on the outer surfaces have considerable freedom to move. For example, the phenolic ring of tyrosine 248 rotates from position A to B to participate in the hydrolytic reaction51 and, as will be demonstrated below, the hydroxyls of four serines in plane 0 can be rotated by surface water to reinforce transient linear hydration order in plane 1.

The Catalytic Reaction Site

The shaded areas in both Top and Front Views outline regions where substrate polypeptide chains bind as they move into the reaction site and prepare for hydrolysis. It is quite amazing that each of the groups in this binding site, as well as peripheral groups, appear to play a role in defining the effectiveness of catalysis. For example, the phenyl group at 279 and the methyl groups of leucine 125 provide a water-ordering lipid barrier to direct the water-ordering aromatic ends of polypeptide chains into the highly ionic reaction site containing a cationic zinc ion, two cationic arginines at 127 and 145 and the glutamate at 270.

Top and Front Views the catalytic reaction binding site of the Carboxypeptidase A enzyme.

It is rather amazing that a zinc ion binds selectively in the reaction site when a multitude of other divalent cations, like magnesium, calcium and iron, might also bind. However, the nitrogen atoms of the two histamines and the oxygen of the glutamate shown in the Front View below the zinc ion are in precisely the proper positions to selectively bind a zinc ion rather than any other ion. Bound tightly in position, zinc holds a single water molecule in a critical position above it.

Likewise, the other groups around the site are in positions which permit water molecules, as well as substrate molecules, to bind in specific positions and be moved in proper directions to perform the hydrolysis reaction rapidly and efficiently. As mentioned before, the hydroxyl groups of the four serines in plane 0 in the right-hand loop and the phenolic group of tyrosine 248 are relatively free to rotate to hydrogen bond with both water and substrates in plane 1. Remember, it was glutamate at 270 which played an important role in the formation of the loop over the aryl binding site and in gathering the cationic arginines into the site. It is almost as if the site were designed by some sort of “Master Plan.”

Reaction Site Hydration

Although the positioning of four serines below hexagonal sites in plane 1 seems almost preconceived, it is likely that this protein, like all of natural molecules, was formed by trial and error based on bonding angles and distances. Polypeptides which formed stable spatial forms under the direction of transiently linearizing water, survived, those which did not, were chewed up by lytic enzymes. It is likely that multiple random genetic changes occurred in polypeptide sequences before the one shown here appeared. The fact that slight differences in polypeptide sequence are found in proteins from different species is evidence that many genetic changes occurred during biomolecular evolution.20

Dielectric transient linear elements of hydration convey a positive charge from the Carboxypeptidase reaction site.

Although water molecules are illustrated in specific positions, they are only probability locations – those on the surface are extremely dynamic and exist only as short elements for an instant. Normally, surfaces which induce order in adjacent water are composed of nonpolar, hydrophobic groups. In this case, the ordering region has aromatic and aliphatic groups at 279 and 125, but the major ordering groups are polar serines which, most likely, induce planar as well as linear covalent order. In this protein, serines on a planar surface provide for linear hydration order. In the feet of penguins, acids and amides in positions of water molecules in planar proteins disrupt transient linear order and prevent water from freezing, even at extremely low temperatures.52 However, as mentioned in the introduction, Dr. Davies and his group at Queen’s University in Canada recently reported that a protein, with as many as 400 water molecules trapped in fixed ordered positions between coils, exhibited antifreeze properties.29 In spite of the fact that a highly-ordered form of water was in contact with surface water, it was not in linear hexagonal forms. Like the trans form of trihydroxy cyclohexane presented previously,39 it disrupted transient linear hydration and ice formation by not providing the correct positioning of polar surface groups.

Substrate Binding

As the anionic end of the polypeptide chain moves into the cationic reaction site, the phenolic oxygen of a terminal tyrosine peptide most likely binds by water bridging to the serines in several alternative positions and then on into binding above arginine 127.

Polypeptide substrates are directed into the binding site by linearly bridging to serine hydroxyls.

When firmly bound in the site, as illustrated on the lower right, the carboxylate ion of tyrosine is bound tightly to arginine 145 with the phenol ring under the loop hydrogen bonded to aspartate 256. (The loop has been removed to reveal the binding.) The carbonyl oxygen of the second peptide is directly above the zinc ion. In the process of binding, the substrate chain has released multiple ordered water molecules to increase local hydration entropy and assist in driving the binding.26

The figure on the lower left illustrates how, with a slight movement of the second peptide carbonyl, it can bond with the oxygen of glutamate 270 to form a critical tetrahedral hydrolysis intermediate.

Substrate Cleavage

The O-C bond in the intermediate shown in Figure 1 below can either break to reverse the process or, as shown in Figure 2, the C-N bond on the same carbon can break. This releases the nitrogen as a free amine, the terminal tyrosine as a free molecule and leaves the carbonyl of the peptide bonded to glutamate 270 as an anhydride. What is not shown is that tyrosine 248, which is shown in Figure 3, hydrogen bonds to the amine of the released tyrosine molecule and escorts it out of the site.

Substrate calalytic cleavage involves a cleavage of the terminal aminoacid and then release of the continuing polypeptide.

Calculations of bond energy suggest that binding in the site puts stress on the bond of the terminal tyrosine which is released on breaking the peptide nitrogen bond but hydrogen bonding to tyrosine 248 also assists in the hydrolysis.51 As the tyrosine molecule leaves the site, five water molecules move in to fill it. As can be seen in Figure 3, one water molecule, w, is held directly above the anhydride by tyrosine 248.

Reaction Detail

In this Front View, once again, we can see how tyrosine 248 most likely is involved in the reaction. Once the terminal polypeptide substrate is bound tightly in the site, as in A, carbonyl 270 forms the tetrahedral intermediate, as in B. When the N-C bond breaks, the phenol on tyrosine 248 rotates around its bond and escorts the free terminal amino acid out from under the loop. Of course, as the amino acid leaves, water moves into the site.

. Detailed mechanism of the catalytic cleavage reaction.

With the one of the oxygens of the anhydride directly over the zinc ion, the water molecule hydrogen-bonded to phenol 248 in Figure D bonds to the carbonyl carbon, forms the free acid and releases glutamate 270 as in E. Once again, the phenol ring of tyrosine 248 most likely escorts the released polypeptide chain from the site.

As indicated in the introductory section of this article, the question of water’s involvement in the assembly and function of proteins has largely been ignored because of the extreme diversity of hydration options and the extremely dynamic nature of water molecules in the liquid state. However, armed with the Transient Linear Hydration Hypothesis and computers capable of handling the thermodynamic effects of rapidly-forming linear elements of hydration, it may be possible to develop more detailed interpretations of vital processes. Now we will look at alpha-chymotrypsin.

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