Polypeptidic backbone modifications

It was said that reduction as well as oxidation of proteins lead to polypeptidic backbone modifications, fragmentation, dimerization or polymerization. Three... 

Transition metals such as iron can catalyze oxidation reactions in aqueous solution, which are known to cause modification of amino acid side chains and damage to polypeptide backbones (see Chapter 1, Section 1.1 Halliwell and Gutteridge, 1984 Kim et al., 1985 Tabor and Richardson, 1987). These reactions can oxidize thiols, create aldehydes and other carbonyls on certain amino acids, and even cleave peptide bonds. The purposeful use of metal-catalyzed oxidation in the study of protein interactions has been done to map interaction surfaces or identify which regions of biomolecules are in contact during specific affinity binding events. 

All the constituent amino acid sidechains in proteins are susceptible to attack by oxidants and free radicals, but some are more vulnerable than others. Thus, exposure of proteins to free radical-generating systems may induce tertiary structural changes as a consequence of modifications to individual amino acid sidechains. As secondary structure is stabilised by hydrogen bonding between peptide groups, interactions of radical species with the polypeptide backbone and interference with the functional groups of the peptide bonds may cause secondary structural modifications. Disruption of the secondary structure may also occur under certain conditions of free radical attack at the a-carbon atom of the peptide bond [20],... 

FIGURE 8.3 A hypothetical series of isomorphous heavy atom derivatives for a crystalline macromolecule, represented here by the polypeptide backbone of rubredoxin. (a) The apo-protein, stripped of its metal ion, provides native structure factors />, shown in vector and waveform on the right (b) the protein with its naturally bound iron atom and FHi, the first derivative structure factor (c) the protein with its iron plus an attached mercury atom, and the resultant structure factor Fm from the double derivative (d) a second multiply substituted derivative formed by attachment of a gold atom to the protein-iron complex. This last derivative is only marginally useful, however, since the reaction with gold also produces a modification in the tertiary structure of the protein (denoted by an arrow). Since this non-isomorphism is equivalent to introducing a nonnative structure factor contribution, the observed F s cannot be properly accounted for, and an erroneous heavy atom contribution / results. This final derivative will yield an inaccurate phase estimate 0v for the native protein. 

Proteins react very efficiently with primary water free radicals, e aq and OH free radicals may react with many targets and hence various forms of modifications are expected of the polypeptidic backbone (dimerizations and polymerizations, fragmentations), and of residues. These reactions have been studied extensively, especially those of OH free radicals because of their importance in biological oxidative disorders (5, 7). 

Protein radiation chemistry has been studied for more than 30 years and a wealth of data has been accumulated. In the solid phase, only modifications of the polypeptidic backbone were shown. They concern the surface. More precisely it seems that weak points are turns and loops. Nothing is known concerrung modifications of residues. The knowledge about radiation chemistry of membrane proteins is also extremely poor. Efforts in this field would be relevant for biology. Let us mention that one of the most important free radical producer systems of living cells is partly buried in a membrane (NADPH oxidase) (see for instance 238). 

Part of the microheterogeneity shown by isoelectric focusing of glycoproteins from the electric organ of the eel Electrophorus electricus is attributable to variations in the content of neuraminic acid. Treatment of the glycoproteins with neuraminidase reduces the number from ten to four. The microheterogeneity is due to post-translational modification of oligosaccharides on a common polypeptide backbone. 

In the NMR conformational study of folded polypeptides, in addition to the well established NMR techniques for small peptides (2-4) one may also rely on the NMR parameters of the polypeptide side chain signals for detection of local conformational transition, intramolecular interactions and chemical modification effects, resulting not only from neighbouring amino acid residues, but also from residues which, while remote in the primary structure, become close to one another by the folding of the polypeptide backbone(5). 

Glycoamine Synthesis. The covalent coupling of amino acid monomers and polypeptide fractions to carbohydrate backbones, previously described (30), was completed using a stationary pH modification (31) of a previously published method (32). 

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