Establishment of Secondary, Tertiary, and Quaternary Structure

Although it is clear how a particular secondary, ter­tiary, and quaternary structure is maintained, how such a specific arrangement is initially achieved is not fully understood.

Cells appear not to possess tem­plates or special enzymes that function in molding a particular three-dimensional shape.

Instead, most polypeptides spontaneously assume their biologically active shape from among the many different alterna­tes that exist.

As we have seen, polypeptide chains are flexible. Thus, free to twist and fold and subject mainly to kinetic and steric constraints, polypeptides can progress through a variety of different shapes, progressively assuming the shape that is energetically most stable.

A polypeptide chain is formed in situ by the sequential addition of individ­ual amino acids beginning at the chain’s N-terminus. Clearly, for many proteins, encoded in the polypep­tide’s primary structure are critical sequences of amino acids that promote the progressive assumption of a specific tertiary structure by the polypeptide as the primary structure is being laid down.

Such pro­gressive folding significantly reduces the total num­ber of possible alternative shapes assumable by a poly­peptide. In other words, once folding of a portion of the polypeptide proceeds to its thermodynamically stable state, certain alternative configurations made possible by continued elongation of the primary struc­ture are no longer kinetically accessible.

For proteins composed of two or more polypeptide chains and possessing quaternary structure, it is likely that the polypeptide subunits randomly encounter one another, automatically orienting themselves and combining in such a manner as to establish the proper (i.e., biologically active) quaternary structure. Some of the experimental evidence supporting these ideas follows.

Dissociated (or protonated) side chains of the acidic and basic amino acids create negative and positive sites along the polypeptide. These sites can be coordinated and stabilized by water molecules, which are also partially polar or by dissolved ions. Conse­quently, segments of a polypeptide containing such charged residues would naturally tend to orient them­selves at the outer surface of the protein where they could interact with the surrounding aqueous environ­ment.

The side chains of other amino acids such as tyrosine, glutamine, and so forth, which are electri­cally neutral on the whole, contain atoms of nitrogen and oxygen that are partially polar due to the differ­ential electrophilia of these atoms.

Therefore, water would also be attracted to these groups, although not to the same degree as fully charged side chains. By in­teracting with electrically charged surface groups, the water molecules minimize the strength of the elec­tric fields surrounding these groups and in so doing stabilize the molecule’s structure.

Amino acids such as leucine and phenylalanine are nonpolar and have hydrophobic side chains. Hydro­phobic groups disturb the haphazard arrangement of water molecules, tending to impart order to the water. Any increase in order makes a system potentially less stable by reducing the system’s entropy. Because entropy tends to be maximized, the hydro­phobic side chains are usually turned away from the surrounding water and face each other internally.

Accordingly, among the many proteins that have been extensively studied, most of the amino acids with polar side chains are at the protein’s outer surface, whereas the amino acids with hydrophobic side chains are either confined to the interior of the molecule or form crevices in its surface so as to have minimal con­tact with the surrounding water and ions.

Anfinsen’s Studies with Ribonuclease:

The notion that, acting in concert, the specific primary structure of a polypeptide and the innate properties of the side chains of its amino acids cause the polypeptide to spontaneously assume its biologically active tertiary structure originated with the studies of the enzyme ribonuclease begun in the 1950s by C. B. Anfinsen; later (1972) Anfinsen received the Nobel Prize for this most definitive work.

Ribonuclease is produced by cells in the pancreas and is then conveyed through ducts to the duodenal section of the small intestine, where the enzyme acts to degrade RNA in ingested food. Ribonuclease contains 124 amino acids, has a molecular weight of about 12,000, and consists of a single polypeptide chain.

Anfinsen identified four di­sulfide bridges in the protein, suggesting that the en­zyme is highly folded (Fig. 4-23). As is the case with nearly all enzymes, the catalytic activity of ribonucle­ase depends on the maintenance of a particular three- dimensional shape. In concentrated solutions of β-mercaptoethanol and urea, the disulfide bridges of the enzyme are broken and the resultant unfolding of the polypeptide is accompanied by a loss of enzyme ac­tivity.

The enzyme is said to be denatured. If the β-mercaptoethanol and urea are removed by dialysis and the denatured ribonuclease reacted with oxygen, the four disulfide bridges re-form, and essentially all of the catalytic activity of the protein is restored (see Fig. 4-23).

Similar observations have been made with other proteins, that is, they are capable of spontane­ously reestablishing their biologically active tertiary structure after having undergone extensive molecular disorganization. In these proteins, the three-dimen­sional structure that is crucial to biological function is directed by the primary structure, and in the appro­priate environment one particular configuration is overwhelmingly favored energetically over other pos­sibilities. Polypeptides possess sufficient flexibility to explore various configurations, progressively assum­ing the most stable of these.

Spontaneous Formation of Quaternary Structure:

Studies of a number of different proteins composed of two or more polypeptide chains support the concept of spontaneous assumption of a specific and functional quaternary structure from the separate subunits. Among those proteins with which the phenomenon is readily demonstrated is the blood protein hemoglobin. Normal adult human hemoglobin, called hemoglobin A, may be represented as

α₂β₂.

This notation indicates that the protein is a tetramer that is, it consists of four polypeptide chains—a pair of alpha chains and a pair of beta chains (Figs. 4-24 and 4-25). If an aqueous solution of hemoglobin is made weakly alkaline or weakly acidic, the hemoglobin tetramers undergo a stepwise dissociation into sub- units, as follows:

As indicated in the above reaction, the dissociation of the tetramer, first into “asymmetric dimers” and then into monomers or subunits, is reversible, that is, when normal pH is restored, fully functional hemoglo­bin molecules are sequentially re-formed. These and similar observations with other proteins support the view that intrinsic properties of the individual sub- units of a protein are sufficient to promote the assumption of the biologically active quaternary structure. In the case of hemoglobin, it appears that the subunits seek out one another in solution and com­bine to form the biologically functional tetramer.