Science in Christian Perspective


The Secondary and Tertiary Structure of Proteins and Its Biological Significance


From: JASA 13 (December 1961): 111-113.

A singular feature of the biological world is the interdependent structure which integrates to give a functional whole. One finds at all levels special arrangement of structure such as to produce the most efficient adjustment to the environment for the biological systems (6, 8, 9, 13). Moderate variation or heterogeneity is permitted the biological structure but gross departure from the norm results in malfunction for the entire system.

The importance of protein structure and its significance to the biological system are best put into perspective by first considering the structural organization of biological entities. Examination of the species of higher organisms of either kingdom reveals that they are comprised of distinct but functionally interdependent organs. Thus, in the case of the plant is found such organs as the root, stem and leaves with each structure functionally related to the well being of the other. Similarly in animals, the various organs are clearly discernible and their relation one to another indisputable. Analysis of a function of the several organs reveals that not only is the location of these organs with respect to one another important but that the geometry of the organ itself is a critical factor for its efficient functioning. Thus, for example, where respiratory activity is high, and large amounts of gas are exchanged, the organ will often assume a spongy, laminar shape as in the case of leaves. Other geometrical designs in organs are such to provide the optimum in functional efficiency.

One can follow this analysis to the level of tissues comprising the different organs. Here again the arrangement of structure is such as to promote the interdependence of the different cell types in the tissue for maximum efficiency. Again, as may be seen upon microscopic examination of an organ such as a leaf, structure and configuration is important in the functioning of the cells of a given tissue.

At the cellular level the orderly array of discrete units is still very much apparent. Thus, we find in the cell such structures as the nucleus and the mitochondria, microsomes and plastids of the cytoplasm. These subcellular units are arranged in such a way as to provide the cell for functional efficiency. Examination of the organization and structure of the individual subcellular particles has also revealed an orderly functional arrangement. For example, in mitochondria the molecules of its component enzymes are ordered in such a way as to accomplish transport of the electrons liberated in oxida-

*Paper presented at the Fifteenth Annual Convention of the American Scientific Affiliation held at Seattle, Washington, August, 1960.

**Dr. Freed is a Professor of Agricultural Chemistry at Oregon State University.

tion with maximum efficiency (13). It has been found that agents of conditions that modify the morphology of these mitochondria materially modify the ability of these subcellular particles to perform their normal functions (15). Such things as the osmotic pressure of the suspending medium (15), ultraviolet light (4), and chemical agents (22) are all capable of inducing serious changes in the ability of the mitochondria to function.

In view of the orderly structural arrangement at the cellular level and above, one would expect to find some order of conformation at the level of the molecules comprising the cell. However, order at the molecular level is not immediately distinguishable visually. It is not surprising, therefore, that our concepts of the structure of proteins in the living cell is only now emerging into a more exact state of knowledge (8, 10, 20). The important relationship of proteins to the living activity of the cell has long been recognized. The ability of many proteins to promote chemical reactions essential to life by serving as catalysts has been the subject of thousands of investigations (6). The key role of proteins in living processes has lead to a determined effort to elucidate the composition and structure of these very important biological materials (6, 10).

The prime function of a catalyst in promoting a reaction is usually to provide a surface at which the reaction occurs. Thus, a catalyst confers on the molecule, by sorption, the ability to become more reactive through a change in its configuration, or a change in the electron density at a particular chemical bond. It is a wellrecognized fact that the composition and structure of the catalyst is of paramount importance in determining which particular chemical reaction it will promote. This specificity of catalysis readies its climax in the enzymes of the living cell (6). It is for this reason that the structure or geometry of surface of these particular substances is of such importance to the biological system. In order for the enzyme to promote a reaction, it is necessary for the enzyme and the reacting molecule to interact to form a complex. The fastidious requirement for certain specific sites by biochemical molecules imposes a stringent requirement for structural specificity on the enzyme that is to catalyze the reaction.

Early study of protein chemistry soon revealed that proteins were composed in large measure of amino adds joined together through an amide or peptide bond. Thus, the amino group (NH,) of one amino acid was linked to the carboxyl group (-COOH) of another amino acid. Since each amino acid contained at least one each of an amino group and carboxyl group, it would be possible to build up high molecular weight polymers of such units. Additional groups such as the sulfhydryl (SH) of certain amino acids provide further functional groups which may react to form chemical bonds in the polymer. The sulfur atoms found in the polypeptide molecule cross link at defined intervals and provide further stability to the polymer.

Initial efforts in protein chemistry were directed at determining the specific amino acid composition of various proteins. It was not long until a considerable body of knowledge was built up regarding the kinds and even the numbers of amino acids found in different proteins. With this information in hand, the next question concerning the protein chemist was to determine the order in which the some 20 amino acids were arranged in the protein molecule. The efforts directed toward elucidating this primary structure Of protein molecules engaged the attention of protein chemists for many years. It wasn't until some of the newer techniques were introduced that it was possible to map the arrangement of amino acids in a protein molecule (6, 10). The very excellent work of Sanger (16) and others (6, 10) resulted in establishing the amino acid sequence of the protein insulin.

Study of the kinetics of enzyme reactions and the physical chemistry of proteins in solutions suggested that enzyme reactions were occurring at the surface of proteins. The biochemical reactions are so specific that this can only be accounted for by the structure of the catalyst. Accordingly, a great deal of attention had been given to elucidating the secondary and tertiary structure of proteins in solution or in biological environment. Detailed consideration of the chains formed by the peptide linkage of the amino acid finally resulted in the suggestion that these chains form spirals or coils known as helices such as are found in springs (6, 19). This suggestion leads to the proper interpretation of X-ray data obtained on proteins and gave a new clue as to the structure of enzymes.

The protein chemists next concerned themselves with the manner in which the peptide chains arranged themselves to form the two major classes of proteins, namely fibrillar and globular proteins. Recent studies have shown that the different peptide chains could arrange themselves in the form of pleated sheets (fibrillar proteins) or in coiled, spherical particles (globular proteins).

Recent work on synthetic polypeptides has revealed how the peptide chain of a globular protein can form the alpha helix which is so characteristic of this class of compounds (19). It is now known that the hydrocarbon in skeleton in such chains orient themselves to form the various helical forms. The helices then are stabilized by the chemical bonding between sulfur atoms in the chain through ionic interactions, and perhaps of greatest consequence, through intramolecular hydrogen bonding (12). Szwarc (19) also points out that the hydrocarbon or hydrophobic portions of such molecules may interact further to produce the stero specific coils found in the protein molecule. Thus, as an amino acid is added to the end of a peptide chain, the carbon skelet ? a of this last amino acid not only interacts with the units just adjacent to it but may also interact with units in the chain removed. in position from it.

Various studies, particularly that of fluorescence and optical rotation, have indicated that portions of the protein molecule are characterized by an orderly or nearly crystalline structure (8). This has been supported also by X-ray data. Viscosity measurements show that in aqueous media these proteins assume a globular shape. The orderly structure found in the proteins is now believed to be due to the interactions of one portion of the molecule with another through establishment of van der Waals forces and hydrogen bonds. The solvent in which the molecule resides is very important in determining the structural features that the molecule will assume. Thus, in aqueous media or in very dilute salt solutions the enzyme molecule assumes the normal globular structure which provides the surface es ential to its catalytic action. However, it is pointed out that in good solvents, that is, those in which the chemical had a high solubility, the molecule tends to produce a random coil structure in which the globular shape is lost and the peptide chain lengthens out (19, 20). In such case, the hydrogen bonds are broken; often indeed the -S-S- bonds of the protein are broken. Such a situation, known as denaturation of the protein, results in a loss of its enzymatic activity. The aqueous media being a poor solvent for the protein molecule allows the material to assume a shape required for enzymatic activity. In this case the coiling of the chain and its folding to produce a globular crystalloid region is due to the intramolecular bonding and the lack of affinity of the hydrocarbon skeleton of the chain and water. In such a situation various functional groups such as a histidine-nitrogen and a carboxyl group or hydroxyl group of a tyrosine are brought into juxtaposition to provide a site at which a reactive molecule might be adsorbed.

A slight modification of the tertiary structure of an enzyme results in a marked change in its catalytic activity (18). Various conditions and agents are capable of bringing about the slight structural modifications with such things as heat (3), ultraviolet light (2, 4, 14, 18), chemical agents (3, 7, 21) and various salts and solutions producing a marked change in the enzymatic activity of a given protein. Conversely it has been found that substrates and coenzymes (16, 22) and such things as fatty acids (3, 5) may stabilize a particular configuration of the enzyme surface thus maintaining its enzymatic or catalytic activity even under conditions designed to destroy it. The author in studying the effect of certain chemical agents on the enzyme amylase found calcium to stabilize the configuration of this enzyme and make it extremely resistant to denaturation whereas univalent ions such as sodium, while giving the same degree of stimulation to the enzyme, would not protect it from attack by the chemical agent.

In the biological system, the structure assumed by the enzymes is of immediate consequence to the respiratory activities of the organism. The possibility of interaction of one protein with another (1), with substrates or with naturally produced chemicals such as hormones (21) provides a means by which the proper configuration is maintained, It has been proposed that enzymes catalyzing certain specific reactions may have their enzymatic properties modified by the presence of certain naturally occurring substances (23). On the one hand the substances may be produced to promote or inhibit the catalytic properties of the enzyme depending on the requirement of the biological system. Thus, the cell would have a built in governor in that by simply modifying the surface geometry of its enzymes it could respond to conditions of its environment or growth requirement. This would be a control in addition to that imposed by concentration of equilibrium.

It has been repeatedly noted that both enzymatic activity and cellular activity of an organism is inhibited by exposure to ionizing radiation or to other agents in its environment. It is clearly established that enzymes (2), mitochondria (4), and organisms may be thus affected. It is implicit in our considerations here that a great deal of this results from modification of the tertiary structure of the proteins. The requirement of a specific structural configuration for enzymatic activity, on the one hand, and the ability of ultraviolet light and other agents to destroy this structure, on the other hand, raises an interesting point with regard to recently suggested schemes (11) of enzyme development on the primordial earth. Although not all authors are in agreement on the conditions that were to have existed at the time (8), most appear to be agreed that ultraviolet radiation and ionizing radiation must have been at a high level. "Primordial protein" existing in a solution, would be thought to be particularly susceptible to having their catalytic powers destroyed by such radiation. It has been suggested that these initial protein-like materials may have been adsorbed on clays or other inorganic surfaces prior to organization of a biological complex. The ability of surfaces to order molecules thus facilitating reaction has been demonstrated (17). However, it would seem that the multifunctional behavior of enzyme systems would require an association of like molecules for further development. It would seem likely that such systems would be most susceptible to the ionizing radiation or other factors of the environment.

Moreover, it has been reckoned t h a t the waters in which such systems may have arisen were deficient in salts. These salts are formed of cations that afford protection of structure to proteins. Such facts as these require of any scheme of protein evolution explanations of how these barriers to enzymatic activity may have been surmounted. Some protection from radiation would be afforded by a sufficient layer of water but diffusion and other types of movement would have sooner or later resulted in these molecules moving to the surface where the possibility of exposure would be increased.

Much remains to be done before complete understanding of the importance of the tertiary structure or geometry of proteins is fully understood. It is not premature to say that the structure assumed by proteins is of utmost consequence to biological systems. We see in this a further means by which sequential chemical events may be controlled in that substrates such as hormones or other chemicals may modify structure, thus increasing or decreasing catalytic activity. Further studies of this problem can only serve to give us a deeper appreciation of the magnificent structural order of the biological world.

(1) Annau, E., Arch. Biochem. and Biophys., 78, 206, 1958.
(2) Aprison, M. H., Arch. Biochem. and Biophys., 78, 260, 1958.
(3 Ballou, G. A., et al., 1. Biol. Chem.,
153, 589, 1944.
(4) Beyer, R. E., Arch. Biochem. Biophys., 79, 269, 1959.
(5) Boyer, P. D., et al., J. Biol. Chem.,
162, 181, 1946.
(6) Dixon, M., and Webb, E. C., Enzymes, Academic Press, New York, 1958.
(7) Duggan, E. L., and Luck, J. M., 1. Biol. Chem., 172, 205, 1948.
(8) Edsall, J. T. and Wyman, J., Biophysical Chemistry,
1, Academic Press, New York, 1958.
(9) Erhret, C. F., Science,
132, 115, 1960.
(10) Fox, S. W., and Foster, J. F., Introduction to Protein Chemistry, John Wiley and Sons, New York, 1957.
(11) Fox, S. W., Science,
132, 200, 1960.
(12) Goodman, M., and Schmitt, E. E., J.A.C.S., 81, 5507, 1959.
(13) Green, D. E., Subcellular Particles, ed. T. Hayashi, Ron ald Press Co., New York, 1959.

(14) Hvidberg, E., et al., Nature, 181, 1338, 1958. (15) Laties, G., Plant Physiology, 28, 557, 1953. (16) Sanger, F., Adv. in Prot. Chem., 7, 1, 1952. (17) Seigel, B. Z., and Seigel, S. M., Nature, 188, 391, 1960. (18) Smillie, L. B., Biochem. Biophys. Acta, 34, 548, 1959.

(19) Szwarc, M., Chem. and Ind., Nov. 29, 1958, p. 1589.

(20) Tanford, C., Symposium on Protein Structure, ed. A. Neuberger, John Wiley and Sons, New York, 1958.

(2 1) Topper, V. J., Maxwell, B.S., and Pesch, L.A., Biochem. Biophys. Acta, 37, 563, 1960.
(22) Ungar, F., and Kadis, S., Nature, 183, 49, 1959.
(23) Zalkin, H., and Tappel, A. L., Arch, Biochem. and Biophys., 88, 113, 1960.