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SURFACE HYDRATION OF POLYPEPTIDES AND PROTEINS

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J. C. Collins, PhD

Dedicated to the late Professors Carl Djerassi and

William S. Johnson of Stanford University


The purpose of this article is to present the concept that the folding and assembly of natural polypeptides into proteins is driven and assisted in direction by the formation of two forms of hydrogen-bonding in surface water. As nascent linear polypeptides emerge from exit tunnels in ribosomes, tetrahedral point-charge hydrogen bonds between surface water and small peptides exchange energy to stabilize the chains. At the same time, water molecules in unstable covalent linear elements of hydrogen-bonding adjacent to hydrophobic surfaces, by spontaneously moving away from those surfaces toward disorder, absorb free-energy from contact peptides and drive them into more orderly stable assemblies. In this article, hydrated conformational forms of polypeptides are used to illustrate how this transition in surface water may drive linear chains into coils, beta-sheets and beta-turns and how it may drive the 42-peptide carboxyl-terminal segment of the Carboxypeptidase-A enzyme into a coil and then into its natural protein structure.

SURFACE HYDRATION OF POLYPEPTIDES AND PROTEINS

You can hover your mouse over illustrations for a brief description

Although proteins form and function only in aqueous media, water is never displayed around them and little attention is directed to the roles it may play in their motions and interactions.1 In the renowned polypeptide-folding experiment performed by Anfinsen in 1961, a ribonuclease protein was denatured by the addition of urea to the aqueous media and then found to be restored to normal function when urea was removed.2 At the time, urea was considered to disrupt hydrogen-bonding within proteins so it was concluded that all of the information required to direct folding was within the polypeptide, that the driving force for assembly was a decrease in internal free energy in the polypeptide and that water was simply the solvent. Although that concept still prevails,3 numerous studies have documented that water produces structuring on surfaces,4 that urea disrupts structuring5 and that water on hydrophobic surfaces most likely drives protein folding.6 In fact, recent studies of water at low temperatures7 suggest that it might be two different forms of hydrogen-bonding between water molecules on surfaces which drive and assist in directing folding and assembly. The purpose of this presentation is to describe these two forms and how they most likely perform those functions.


Historically, each water molecule in the liquid state has been viewed as hydrogen-bonded in tetrahedral configurations to three or four other water molecules by point charges on their surfaces.8 In proteins, water molecules bridge between polar and ionic atoms at a variety of distances and angles to stabilize the molecules in their thermodynamically-stable states.3 Hydrogen bonding between water molecules in the liquid state is weak, 1.3 to 2.8 kcal/mole, and lasts only about 10-12 seconds but ties the molecules together in coordinated clusters.8 However, in 2004, neutron bombardment at the Stanford Synchrotron Radiation Laboratory provided evidence that, at any instant, the largest structural unit in liquid water is a “trimer,” with two water molecules hydrogen bonded to a central molecule.9  Molecular orbital calculations in the 60’s and 70’s forecast that such a trimer (with 2.76 Angstroms between the molecules) would be the most stable structural unit in liquid water10 and, in 1972, X-ray scattering from the surface of liquid water at 25oC revealed the presence of both trimers and tetramers, again with 2.76A between the molecules.11 Trimers and tetramers form spontaneously but are unstable and last only about 10-12 seconds, a million millionth of a second.12


When the trimer was first reported, most scientific attention was directed to crystalline proteins and there was a debate regarding the nature of the hydrogen bond.8,10  However, in 1999, Dr. Isaacs at Bell Laboratories concluded, based on detailed X-ray analysis, that water-to-water hydrogen-bonding in ice is “covalent”- similar to the bonding between carbon atoms with the electron clouds of adjacent water molecules encompassing a central proton.13 Covalent hydrogen bonding is relatively ridged in the various forms of ice with energies ranging from 4.7 to 8.2 kcal/mole, but is unstable above 0oC with half-lives of about 10-12 seconds.13 Further evidence for this second form of hydrogen-bonding between water molecules was provided in 2003 by Professor Stanley at Boston University who, as a consequence of a detailed study of the properties of liquid water, concluded that water in the liquid state and on surfaces is composed of two different density-forms of water molecules.7


If liquid water is carefully cooled to -30oC, it turns into a glassy fluid containing linear elements and clusters but no ice.7,14 However, if cooled down to -40oC, crystallization occurs immediately to produce a form of ice called “cubic” in which all of the molecules are covalently bonded together in linear elements, 2.75 Angstroms between the molecules.14 Cubic ice, like the trimer, is formed kinetically by the linear overlap of orbitals15 but is unstable and isomerizes immediately at 0oC into the more stable hexagonal form of ice in which some of the elements are not linear and the molecules are 2.75 to 2.84 Angstroms apart.14 It is important to realize that water molecules have extremely high kinetic energy with a low probability of spontaneously forming covalent two and three-dimensional forms, even below 0oC.8  However, if liquid water is placed in contact with a surface in which the atoms are in same regular hexagonal arrangements as they are on the surface of ice, like those of oil molecules or iodine crystals, water molecules are drawn into hexagonal positions and crystallization occurs immediately at 0oC.8  If even a trace of oil is on the surface of water, super-cooling is impossible because the ends of oil molecules in contact with water assemble in the same hexagonal arrangements as water molecules in ice.16


Water in contact with non-hydrogen-bonding hydrophobic surfaces, like those of oil and lipid molecules, displays the nuclear magnetic resonance doublet peaks of ice, not the singlets of liquid water17 and molecular orbital calculations indicate that linear elements of five and six hydrogen-bonded water molecules form in particular orientations on such surfaces.18 Although half-lives of these linear elements of hydration on hydrophobic surfaces are too brief to permit visualization, ultra-high-speed crystallographic analysis of water on the solid planar surface of graphite by the late Professor Zewail and his group at Caltech, revealed that it is present as layers of hexagonally-bonded linear elements with cubic patterning between the layers – similar to cubic ice.19


Although covalent trimers and linear elements which form on hydrophobic surfaces last no  more than 10-11 seconds,20 they must fill voids and bind between polar and charged atoms as polypeptide chains transition from one thermodynamically-stable state to another. As linear elements between hydrophobic surfaces and charged atoms increase and decrease in length by admitting and releasing single water molecules, polypeptides must move in quantized steps to assemble and perform vital functions.23 In fact, on broad planar hydrophobic surfaces, covalent bonding might well propagate the formation of time-dependent planar sheets and hydration shells with cubic layering4,6 - the same as on the surface of graphite.19


However, the formation of relatively-ridged covalent linear elements on surfaces may not only be responsible for the orderly assembly of proteins but for spontaneity as well. As a water molecule approaches a hydrophobic surface and forms a covalent hydrogen bond, it loses 4 to 5 kcal/mole of quantized energy to adjacent water molecules.7,21  However, as the bonds break and the water molecules return to the more random point-charge state, similar units of energy are absorbed from surface molecules and they are driven toward lower energy and higher order.22 For example, oil molecules, which can twist and turn and bend in multiple ways in the liquid state, lose energy and entropy and are forced to align as linear segments in layers in contact with water. As water molecules escape from the interface, they absorb energy and increase in freedom and motion while oil molecules lose energy and are restricted in motion to lateral motions and rotations around their axes.16


Water molecules are unique in that their small size and highly-reversible hydrogen-bonding property permits them to move rapidly back and forth between covalent and point-charge bonding. By rapidly forming and degrading on ordering surfaces, covalent linear elements of hydration not only transfer units of energy from more massive slower-moving molecules to surface water,22 they move in quantized steps.23 It is this unidirectional transfer of quantized units of energy from biomolecules to water which most likely drives (and possibly directs) the spontaneous folding and assembly of natural polypeptides into functional proteins. By obeying the Second Law of Thermodynamics and moving spontaneously from order toward disorder, water molecules in transient linear elements of surface hydration move natural molecules in the opposite, non-spontaneous direction. In 1937, it was Erwin Schrodinger, in his little book “What is Life?” who concluded that it was this unidirectional transfer of energy which drove molecular evolution from disorder toward order - it was water which produced the order for life.24


Actually, the spontaneous movement of polypeptides into functional proteins involves dehydration.6 As polypeptides emerge from ribosomes, hydrophobic surfaces of specific dimensions, which become coated by covalent linear elements of hydration of similar dimensions, search for complimentary surfaces with which to assemble and release covalent water from both surfaces. By continually releasing ordering elements of water, hydrophobic surfaces form the central cores of proteins.22 When final proteins are produced, central regions are dehydrated with peptides which provided hydration order during assembly on the inside and peptides with amides and  ions in their side chains on the outside, hydrogen-bonding with surface water in multiple orientations to increase mobility, stability and solubility.6,8 In fact, recent studies suggest that exit tunnels in ribosomes, through which newly-synthesized polypeptides must pass, most likely function like chaperone proteins to direct water into ordering regions of polypeptides and assist them to fold into coils before they are released into surface water.25 Although, outer surfaces of most finished proteins do not reflect cubic geometry, they often display external structures which permit them to assemble into complexes with each other and other proteins in a variety of symmetries.3


Clearly, quantized transient linearization in surface water must play a dominant role in filling voids and stabilizing spaces in intermediate states in the formation of functional proteins. However, they also must play a critical role in the transfer of charges between polar and ionic atoms on surfaces of proteins as polypeptide chains move from one position to another.26  By forming as covalent water-to-water dielectric linear elements between ions, they permit proton tunneling to generate counter ions and lower charge potentials.27 By transiently forming in enzyme and receptor binding sites in their open forms, they provide quantized spaces for substrate and regulator molecules to bind and perform functions. NMR analyses indicate that water on smooth muscle and collagen fibers is in particular orientations29 and molecular analyses indicate that the phosphate head-groups of lecithin/cholesterol complexes which compose the inner surfaces of large axons of nerve fibers are the same distances apart as covalently hydrogen-bonded linear elements of water molecules. During depolarizations, with high charge-potentials between nerve endings and nodes, positive pulses most likely tunnel through extended linear elements of hydration at extremely high speeds with almost no loss in energy.30 If our nerves were filled with metal rather than water, we would be combusted by the resistance. Nanotechnology today is searching for superconductivity in electrons - nature has already found it in the protons of transient linear elements of water in nerve cells.


Indeed, it is unfortunate that water is never displayed in biomolecular structures. For example, the classical structure of DNA is never viewed as hydrated but the X-ray crystallographic pattern, which was used by Watson and Crick to develop their helical model, was obtained by Rosalind Franklin by spraying a crystalline sample with water.31 Only when DNA is hydrated with at least 13 water molecules per base-pair, does it exist in the uniform helical coil that has become the symbol of modern molecular biology.32 Only when spaces between phosphate oxygens on opposite sides of the wide groove are transiently-bridged by linear elements of six-to-seven covalently-hydrogen-bonded water molecules to delocalize the charge and the narrow groove by three-to-four water molecules, are filaments of double-helix DNA stabilized as they oscillate and move through their various functional states. In fact, transient linear hydration is so dominant around DNA to stabilize its high negative charge that the water is referred to as “ice-like” and spherically-hydrated sodium ions are held out away from it by several layers of water molecules.32,33


Indeed, it is unfortunate that chemists, biologists and the public at large are denied the truth that it is surface water which plays a critical role in the structures and functions of natural molecules. The fundamental problem is that the kinetic formation of transient quantized covalent linear elements of surface hydration must be accepted as one of the primary mechanisms of energy and spatial control within living cells.13,15,23 For the past century, chemists have used the concept of kinetic control and quantized units of covalent structure to successful devised synthetic pathways to complex natural molecules;34 there is no reason why the same fundamental principles of preferred transient linear hydrogen-bonding cannot be used to interpret the role of surface water in transitions of natural molecules from one stable conformation to another.


The purpose of this brief article is to provide an introduction to the use of theoretical principles of covalent hydrogen-bonding in the standard linear 4.5-Angstrom trimer and related transient linear elements of hydration to derive hypothetical conformational interpretations of hydration-stabilized forms of polypeptides as they transition from linear states to coils, beta-sheets and beta-turns.35 Since surface water is dynamic and continually shifting from one state to another, there is no way to calculate precise energy changes.36 However, as you will see from the proposed intermediates, there is a continual decrease in linear ordering in surface hydration as folding and assembly proceed. Polypeptide side chains are displayed in specific extended low-energy conformations realizing that most of them are dynamic and move relatively freely from one low-energy conformation to another in aqueous solution but are confined to more specific conformations in finished proteins.36 Finally, the folding of the terminal 42 peptides at the acid end of the Carboxypeptidase-A polypeptide is presented as an example of how hydration principles derived above can be used to interpret its folding into a thermodynamically-stable natural protein conformation.37


Within the past few years, an increasing number of studies have provided evidence that surface water and water at sub-zero temperatures display the properties of a quantized media,23 and, recently, the quantum mechanical property of entanglement has been revealed as a property of  the protons within water molecules.38 Undoubtedly, these properties of quantization will permit the development of entirely new concepts regarding the role of water in life processes. The question is: When is the field of molecular biology going to realize that it is the properties of surface water, which Erwin Schrodinger proposed as bringing forth life, going to be recognized as providing order and spontaneity within the living cell and in biomolecular evolution.


For more information on the author, the quantization of surface hydration, the assembly of proteins, the hydration of receptor sites and the possible role of water in natural molecular evolution, check out www.linearwater.com and www.molecularcreation.com.   

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