Site-Specific Modification of Proteins

As a proof-of-principle, this method has been used to generate a dual-labeled Rab7 GTPase. To generate Rab7 with an N-terminal cysteine, a tobacco etch virus 

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Figure 4 Strategies for the application of CoA analogues. For in vitro studies appropriate analogues are prepared from a suitable precursor, purified, and characterized if necessary, and then normally used as mechanism-based probes and inhibitors (panel A). When CoA analogues are used for the in vitro site-specific modification of proteins by transfer of a reporter label to a carrier protein module using a PPTase enzyme, the analogue can be prepared and used in situ (panel B). In vivo reporter labeling is made possible when cells are provided with suitably modified pantothenamide precursors, which are subsequently transformed into the respective CoA analogues and transferred a carrier protein by the cell s native CoA biosynthesis (CoaADE) and PPTase enzymes. The labeled protein can be recovered from the cells by cell lysis.

Fusion Cys-tag for Site-Specific Modification of Targeting Proteins... 

The principle advantage of the physical labeling method is the possibility of receiving direct information about the structure, mobility and local micropolarity of certain parts of a molecular object of any molecular mass. Developments in synthetic chemistry, biochemistry and site-directed mutagenesis have provided researchers with a wide assortment of labels and probes, and have paved the way for the specific modification of protein function groups, including enzyme active sites. 

The site-specific modification of enzymes with a single electron-relay group located near to the redox cofactor and providing efficient electrical contact with the conductive support has been achieved by the reconstitution of enzymes with cofactors covalently linked to redox groups. Affinity interactions between enzymes and their cofactors at the electrode interface can allow the efficient electrical contacting of aligned proteins. 

Site-specific modification of native proteins with group-specific reagents... 

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-... 

The unexpected specificity which can be achieved with functional group modification reagents is an apparent consequence of the native protein s ability to impose a unique chemical environment on a given amino acid under a given set of experimental conditions. It is important to emphasize that the site-specific modification of a protein is a kinetic phenomenon and selective modifications result from the ability of the protein to alter the reaction rate of a single residue under one clearly defined condition of pH, ionic strength and temperature. For example, it is entirely possible that if, at pH 7.0, one lysine residue is substantially more reactive than either free lysine or other lysine residues in the protein it may well be less reactive than these at pH 9.0. 

Two factors are chiefly, but not exclusively, responsible for the fact that, under certain conditions, amino acids in native proteins react more rapidly than free amino acids in solution. The first and most general is the capacity of proteins to bind modification reagents at or near the functional groups of amino acid residues in orientations favorable to reaction. The reversible binary complexes formed between proteins and modification reagents prior to reaction are analogous to enzyme-substrate complexes. As a result, most site-specific modifications of native proteins probably proceed by the scheme summarized in eq. 

Circular dichroism (CD) has played an important role in our studies on the modification of enzymes and hormones with Co(III). The objective of these studies has been to incorporate selectively substitution inert metal ions at specifically modified sites in proteins as probes of biological function. Significant information concerning the catalytic mechanism of carboxypeptidase A (CPA) (1) has been obtained from a site specific modification of tyrosine 248 with Co(III) (2). The method developed for CPA has been extended to other enzymes and hormones in order to devg op an improved method for incorporating stable radioisotopes t Co) into proteins. The substitution-inertness of Co(III) provides the necessary stability in these derivatives (3). 

The application of site-specific modification of enzymes and other proteins has become increasingly common, and a wide range of chemical classes is available from which to select or design a reagent for exploring a particular enzyme. Structural similarity to the natural ligand is always desirable to ensure target sped-... 

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