Principles of Biochemistry Volume 1 - A. Lehninger 1985

Biomolecules
Globular Proteins: Structure and Function of Hemoglobin
The amino acid sequence of a protein determines its tertiary structure

As we have already seen from our Structure/133.html">Discussion of the α-Helix and β-conformation, the Introduction/11.html">Secondary structure of a polypeptide chain, which describes the spatial arrangement of adjacent amino acid residues, is determined by its Amino Acid Sequence. The α-helix and β-conformation arise spontaneously and remain stable only if The sequence of adjacent amino acid residues in the polypeptide chain has an appropriate sequence of R-groups. The tertiary Structure of Globular Proteins is also determined by their amino acid sequence. However, while secondary structure is determined by the sequence of R-groups in nearby Regions of the chain (i.e., short-range order), tertiary structure depends on The amino acid sequence of widely separated regions of the chain (i.e., long-range order). The formation of bends in the polypeptide chain, as well as the direction and angle of the chain's turn in these bends, are determined by the number and position of specific amino acid residues, such as Proline, Threonine, and Serine, which promote bend formation. Furthermore, as we will see later, the loops of a tightly folded polypeptide chain maintain their characteristic spatial arrangement due to various interactions between the R-groups of adjacent loops.

Many invariant amino acid residues of homologous proteins—that is, residues that are always present at specific positions in polypeptide chains regardless of the species from which the protein is obtained—most likely occupy the structurally most critical positions in the polypeptide chain. Some invariant residues are found near or within the bends of the chain, while others, such as cystine residues, are located at sites where cross-links form between closely positioned loops of the tertiary structure. A number of invariant amino acid residues occupy strictly defined positions in the catalytic centers of Enzymes or at the binding sites of prosthetic groups, such as the heme group in cytochrome c.

However, the most convincing evidence that the Tertiary Structure of a globular protein is determined by its amino acid sequence comes from experiments showing that the Denaturation of certain proteins is a reversible process. In most Globular proteins, heating or exposure to extreme pH values leads to the unfolding of chains and loss of biological activity without breaking the covalent bonds of the polypeptide backbone. For many years, Protein Denaturation was considered irreversible; for example, egg white protein coagulated by boiling does not return to its original soluble state upon cooling. However, it was discovered that for some globular proteins denatured by heat or extreme pH, the native structure and biological activity are restored upon slow cooling of the protein solution or a gradual return of its pH to normal; this process is called renaturation.

A classic example of renaturation is that of Ribonuclease, a single-chain protein with four intrachain Disulfide Bonds. Crystalline ribonuclease can be denatured by Treatment with a concentrated urea solution in the presence of a reducing agent, which cleaves the disulfide bonds of the four cystine residues to form eight Cysteine residues. Under these conditions, the polypeptide chain completely unfolds, forming many random loops, and the enzyme loses its catalytic activity (Fig. 8-8). If we now place the solution of denatured ribonuclease into a dialysis bag (p. 144) and submerge it in Water, the low-molecular-weight substances (urea and the reducing agent) will diffuse out of the ribonuclease solution into the surrounding medium. As these substances are gradually removed, the randomly coiled denatured ribonuclease will spontaneously and slowly return to its Native State, acquiring its characteristic correct three-dimensional tertiary structure with full restoration of catalytic activity (Fig. 8-8). This experiment proves that the information required for the proper folding of the ribonuclease polypeptide chain is encoded in the Primary Structure of the polypeptide chain, i.e., in its amino acid sequence.

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Fig. 8-8. Renaturation of unfolded (denatured) ribonuclease with the reformation of correctly positioned disulfide cross-links. The addition of urea disrupts Hydrogen Bonds in the ribonuclease molecule, and subsequent Treatment of the protein with mercaptoethanol (CH₃CH₂SH) reduces and thereby cleaves the disulfide bonds of the four cystine residues, converting them into eight cysteine residues.

In the course of the same experiment, further Evidence of the precision of ribonuclease folding during its renaturation was obtained. It turned out that the eight cysteine residues formed by the reduction of cystine residues in the fully unfolded ribonuclease are gradually oxidized by atmospheric oxygen, resulting in the formation of four intrachain disulfide bonds in exactly the same positions as in the original native ribonuclease. This is a remarkable phenomenon. A random combination of eight cysteine residues to form cystine residues could theoretically yield 105 different variants; however, during renaturation, only a single, specific set of disulfide cross-links characteristic of native ribonuclease is formed (Fig. 8-8). Thus, the polypeptide chain of denatured ribonuclease folds with great precision, leading to the formation of a unique, biologically active conformation and preventing the formation of any "incorrect" conformation. This classic experiment, performed by Christian Anfinsen in the 1950s, proved that the amino acid sequence of a polypeptide chain contains all the information necessary for the chain to fold into its native three-dimensional structure.