Amino acid side chains, modification

Furthermore, it should be mentioned that some of the amino acid side-chain modifications are actually reversible, including thiolation and disulfide bond formation, whereas others are irreversible, like methionine sulfone formation or nitration. 

Oxidative amino acid side-chain modifications do not result in a stable end product of the oxidation process, but very often highly reactive intermediates are formed. These include chemically reactive groups, like ketones and aldehydes, or the formation of protein hydroperoxides. The presence of such protein hydroperoxides leads to a process called protein peroxidation. Here secondary reactions occur if the protein hydroperoxide decomposes and initiates further oxidative reactions, again forming oxidized protein forms. 

Regulation of biological functions can be achieved via catalytic and binding activities of cellular proteins. In recent years the potential of peptide bond CTI for a switch-like control of protein function beside amino acid side-chain modification or drastically reorientation of a whole polypeptide chains became apparent [147]. 

FIGURE 15.2 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The —OH on the interconvertible enzyme represents an —OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group. 

Evolution has provided the cell with a repertoire of 20 amino acids to build proteins. The diversity of amino acid side chain properties is enormous, yet many additional functional groups have been selectively chosen to be covalently attached to side chains and this further increases the unique properties of proteins. Diese additional groups play a regulatory role allowing the cell to respond to changing cellular conditions and events. Known covalent modifications of proteins now include phosphorylation, methylation, acetylation, ubi-quitylation, hydroxylation, uridylylation and glycosyl-ation, among many others. Intense study in this field has shown the addition of a phosphate moiety to a protein... 

The protein was purified by a dialysis procedure, denatured and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting indicated that the protein of interest consisted of two components, one of which increased in concentration as the purification proceeded. The authors initially suggested that this could be due to the presence of a number of species produced by modification of the amino acid side-chains, for example, by glyco-sylation, or by modification of the C- or N- terminus. 

An alternative to modifying the functional group attached to fibrils is to utilise the chemistry present in the amino acid side chains. Furthermore, as peptides often undergo specific modification by enzymes in vivo, these could be harnessed for synthetic purposes. Qll (Ac-QQKFQFQFEQQ-Am, a fibril-forming peptide based on Pi 1-2), was coupled to lysine-based molecules by treatment with an enzyme (tissue transglutaminase, TGase) which results in a reaction between lysine and glutamine side chains [72] (Fig. 32). 

The modification of amino acids in proteins and peptides by oxidative processes plays a major role in the development of disease and in aging (Halliwell and Gutteridge, 1989, 1990 Kim et al., 1985 Tabor and Richardson, 1987 Stadtman, 1992). Tissue damage through free radical oxidation is known to cause various cancers, neurological degenerative conditions, pulmonary problems, inflammation, cardiovascular disease, and a host of other problems. Oxidation of protein structures can alter activity, inhibit normal protein interactions, modify amino acid side chains, cleave peptide bonds, and even cause crosslinks to form between proteins. 

The formation of an aldehyde group on a macromolecule can produce an extremely useful derivative for subsequent modification or conjugation reactions. In their native state, proteins, peptides, nucleic acids, and oligonucleotides contain no naturally occurring aldehyde residues. There are no aldehydes on amino acid side chains, none introduced by post-translational modifications, and no formyl groups on any of the bases or sugars of DNA and RNA. To create reactive aldehydes at specific locations within these molecules opens the possibility of directing modification reactions toward discrete sites within the macromolecule. 

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. 

Table 1 Posttranslational modifications by amino acid side chain modified ...

Cysteine is the most frequently used residue for selective chemical modification of proteins due to its relatively low abundance in proteins and the increased nucleophilicity of the thiol group relative to other natural amino acid side chains. The intrinsic selectivity is low unless no cysteine is present or unless all unwanted cysteines... 

As described for lEC, elution is done by a stepwise or a continuous change of buffer composition. The mildest elution buffer is an aqueous buffer with low ionic strength, e.g., 20 mm Tris-HCl. If it is not successful, desorb with a chaotropic solvent, e.g., 2 M potassium rhodanide (thiocyanate), 2.5 M guanidinium hydrochloride, up to 7 M urea, or with increasing concentrations of methanol or acetonitrile. Especially the use of rhodanide or urea may be accompanied by a chemical modification of amino acid side chains, which disturbs amino acid analysis. 

Many of radioactive isotopes are very useful for the following biochemical processes (Table 6.1). The radioactive label is introduced into macromolecules, especially proteins, either during biosynthesis, e.g., during translation in the presence of S-methionine, or enzymatically, e.g., by use of P-labeled ATP during protein phosphorylation by protein kinases, or chemically by modification of amino acid side chains. Examples for reagents used in chemical radiolabeling of proteins are given in Table 6.2. 

Besides these modifications, certain proteins can acquire prosthetic groups (e.g., hemes, flavins, iron-sulfur centers, and others). The prosthetic groups may be attached covalently (usually to amino acid side chains) in some cases, noncovalently... 

Each type of histone has variant forms, because certain amino acid side chains are enzymatically modified by methylation, ADP-ribosylation, phosphorylation, gly-cosylation, or acetylation. Such modifications affect the net electric charge, shape, and other properties of histones, as well as the structural and functional properties of the chromatin, and they play a role in the regulation of transcription (Chapter 28). 

Covalent modification by phosphorylation and dephosphorylation of hydroxyl groups on amino acid side chains. 

The presence of O2 causes an increase in the destruction of amino acids and in the amount of amide nitrogen formed and also influences the modification of amino acid side chains. Thus, a major product from PGA irradiated in 02 is aspartic acid, which is formed only in traces in the absence of O2. The inversion of glutamate residues is reduced in O2. 

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