Site-specific chemical modifications

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

The site-specific chemical modification of post-translation... 

Yem, A.W. et al. (1992) Site-specific chemical modification of interleukin lb by Acrylodan at cysteine 8 and lysine 103./. Biol. Chem. 267, 3122. 

Tsutsumi, Y., M. Onda, S. Nagata, B. Lee, R.J. Kreitman, and I. Pastan, Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity. Proc Natl Acad Sci USA,... 

Pennington, M. W. (1994) Site-specific chemical modification. In Peptide Synthesis Protocols (Pennington, M. W. and Dunn, B. M., eds.), Humana Press Totowa, NJ, p. 173. 

When the crystal structure of a protein is not available, other techniques can be employed to identify the amino acids that are involved in its structure and function. Commonly used techniques include chemical cross-linking, site-specific chemical modifications, and mutagenesis. Chemical modifications of Met residues using oxidizing agents such as hydrogen peroxide, t-butyl hydroperoxide, chloramine T, and sodium periodate have been useful in identifying structure and function relationships in many proteins (1-7). 

Puthenveetil S, Whitby L, Ren J, et al. Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification. Nucleic Acids Res. 2006 34 4900-4911. 

RL Lundblad, RA Bradshaw. Applications of site-specific chemical modification in the manufacture of biopharmaceuticals. I. An overview. Biotechnol Appl Bio-chem 26 143-151, 1997. 

Tawfik, D. S., Chap, R., Eshhar, Z., and Green, B. S. (1994). pH on-off switching of antibody-hapten binding by site-specific chemical modification of tyrosine. Protein Eng. 7,431-434. 

Concur, D. W., Hill, H. A. O., Moore, G. R., Whitford, D., Williams, R. J. P., The Modulation of Cytochrome C Electron Self-Exchange by Site-Specific Chemical Modification and Anion Binding , FEBS Utt. 206 (1986) 15-19. 

Site-specific chemical modifications For examples, methyl esterification that adds 14m.u. for each carboxyl group (i.e. side chains of Asp, Glu or C-terminus in the peptide) iodination that adds 126m.u. for each Tyr. 

M.W. Pennington, Site-specific chemical modification procedures, in M. W. Pennington, and B.M. Dunn, eds.. Methods in Molecular Biology, Peptide Synthesis Protocols, Humana... 

Wu P, Shui W, Carlson BL, Hu N, Rabuka D, Lee J, Bertozzi CR (2009) Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag. Proc Natl Acad Sci USA 106 3(X)0-3(X)5... 

Flavin-containing mitochondrial MAO-A and MAO-B catalyze the oxidative deamination of neurotransmitters, such as dopamine, serotonin, and norepinephrine in the central nervous system and peripheral tissues. The enzymes share 73% sequence homology and follow the same kinetic and chemical mechanism but have different substrate and inhibitor specificities. Chemical modification experiments provide evidence that a histidine residue is essential for the catalysis. There is also strong evidence that two cysteine residues are present in the active site of MAO. 

Group-specific chemical modification remains a useful method for studies of structure-function relationships in protein molecules, although unambiguous identification of essential amino acid residues and elucidation of their function are nowadays accomplished mainly by X-ray crystallography and site-directed mutagenesis. Chemical modifications... 

Although the total synthesis of a protein allows complete control over the structure, including posttranslational modifications and introduction of labels at desired sites in the sequence, it is still a major undertaking for which most laboratories whose main interest is in the biology of their target proteins are not equipped. In certain cases, for example when the site of introduction of a specific chemical modification is near the C-terminus, a combination of molecular biological and chemical methods has proved to be very powerful. 

The site-specific modification of native proteins is not one of the routine procedures in protein chemistry. It cannot be placed in the same category as end-group labelling or determination of amino acid composition and sequence. The specific chemical modification of a native protein can never be guaranteed because the reactivity of amino acids in a native protein is rarely predictable even if the three-dimensional structure of the protein is known. Unusual pK s of side chains, steric and solvent effects and the proximity of the amino acid residue to a ligand-binding site all influence its reactivity, frequently in opposite directions. However certain well-defined avenues of in-... 

Such enzymatic approaches have limitations in the scope of manipulation of protein stmcture, and therefore are not suited for the preparation of proteins with different hpid moieties at multiple sites and/or with non-natural groups. Moreover, not all lipid transfer enzymes are readily recombinantly available. Chemical protein ligation methods have been developed in the past few years. These methods allow for site-specific lipid modifications of a protein and production in large quantities for cellular, biochemical and biophysical analyses (Table 2). 

Yin,J., Liu, F., Li, X., Walsh, C. T. (2004). Labeling proteins with small molecules by site-specific posttranslational modification. Journal of the American Chemical Society, 126, 7754-7755. 

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