Principles of Protein Structure - G. Schulz 1982

Protein Evolution
Protein Specialization
Criteria for the Fixation of Amino Acid Substitutions in Proteins

A detailed biography of a protein is essential for evaluating the Biological Significance of a given residue. As demonstrated in the previous example, the fixation of Amino Acid Substitutions depends almost entirely on the Biological Role of the corresponding amino acid. However, assessing this role is quite difficult because it may be associated not only with a specific protein function, such as the catalytic action of an enzyme, but with all other interactions of the protein within the Organism throughout its «life cycle» from the activation of the corresponding Gene to the degradation of the polypeptide. And although such detailed protein «biographies» are not yet available, some General Remarks can nevertheless be made*.

Residues on the protein surface are substituted more frequently than internal ones. As shown in Fig. 7.1, b, the frequency of amino acid residue substitutions in a given protein strongly depends on its position in the polypeptide chain and its Location within the three-dimensional Structure. As a rule, surface residues are replaced more often than interior ones. Since outer residues are less critical for protein stability than internal ones, this rule reflects The Importance of maintaining stability for protein function. Exceptions include surface residues that are directly involved in protein function, such as residues forming catalytically active enzyme centers, residues in binding sites for substrates, Cofactors, prosthetic groups, allosteric effectors, or macromolecule-binding sites. For instance, cytochrome c, which interacts with other macromolecules and attaches a prosthetic group, has practically no inactive regions on its surface. This explains the low frequency of fixed Mutations in the cytochrome c molecule (Table 9.1).

The Effect of Amino acid substitutions in the interior of a protein is often compensated by other substitutions. Since Globular Proteins are packed just as densely as crystals of organic molecules (Sect. 1.6), the replacement of internal residues entails a shift in the arrangement of neighboring residues, which typically leads to a decrease in stability. Therefore, substitutions in the interior of a protein occur relatively rarely and in many cases are accompanied by at least one other replacement. Examples of such mutually compensating substitutions have been discovered in ribonucleases [67]. A case of very subtle internal compensation was noted in two distantly related Serine proteases: the internal cluster of Chymotrypsin, composed of Trp-29, Ser-45, Val-53, Val-200, Leu-209, Val-210, Ile-212, is converted in Elastase to Ser-29, Thr-45, Met-53, His-200, Val-209, His-210, and Val-212 without any major changes to the polypeptide backbone [490].

* These generalizations apply not only to proteins operating under normal conditions, but also to those subjected to extreme conditions of heat [491] or hydrostatic pressure [492], such as proteins from thermophilic Bacteria and abyssal (deep-sea) fish, respectively. Apparently, adaptation to these extreme conditions requires only minor changes in Amino acid sequences.

Substitutions of such residues occur more frequently than other replacements. The requirement to preserve function imposes constraints on the frequency of allowable substitutions at a given position in the polypeptide chain. Apparently, many Functions are least disrupted by conservative substitutions, i.e., replacements by similar residues. In this regard, the size, shape, flexibility, and charge of the side chain, as well as its hydrogen-bonding capacity, are all important. For example, the Lys → Arg substitution preserves a mobile positively charged side chain, whereas the Ile → Leu substitution maintains a relatively bulky nonpolar side chain. THE PRINCIPLE OF conservative substitutions was used in Sect. 1.6 to determine the empirical similarity of Amino Acids based on observed frequencies of occurrence (Table 1.2).

Abnormal Hemoglobins illustrate the potential consequences of random mutations. However, even conservative substitutions can lead to severe consequences, as discovered in the case of abnormal Hemoglobin Sydney [493], which contains Ala instead of Val at position 67 of the ß-chain. The replacement of two methyl groups by hydrogen atoms disrupts the heme pocket and significantly decreases protein stability [494] and, consequently, the Stability of the erythrocyte.

Non-conservative substitutions, such as the Introduction of a negative charge into a hydrophobic interior, are incompatible with the stability of the native protein conformation because the resulting Free energy loss is on the order of 10 kcal/mol, which approaches the value of ∆G_total for the folded-to-unfolded chain transition (Sect. 8.1). An example is abnormal hemoglobin Vienna [494], in which the Tyr-130 → Asp substitution in the α-chain leads to major conformational rearrangements, resulting in the negative charge being compensated by a positive one.

Both described abnormal hemoglobins cause severe anemia, thereby demonstrating the consequences of random mutations. Obviously, such mutations will never become fixed.

Class="center">Table 9.2 Mutation probability matrix over an evolutionary pathway of 2 PAM [20]^a

The Amino acids are arranged in the same order as in Table 1.2. All values are multiplied by 10,000. An element of this matrix, m(i, j), represents the probability that an amino acid in Column i will be replaced by an amino acid in row j over an evolutionary interval of 2 PAM, i.e., 2 accepted point mutations per 100 amino acids. Thus, the probability that Ala will be replaced by Ser is 0.0059, and Ser by Ala is 0.0099. The sum of the values in each column is 1.0. The sum of the values in a row represents the «growth factor per 2 PAM» for the corresponding amino acid residue, and ranges from 0.9737 (for Met) to 1.0188 (for Ala and Val).