Cysteine residues, labeling

In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. Cysteine residues in proteins, for example, react with one another to form disulfide species and also react with a number of reagents, including maleimides (typically A ethylmaleimide), as shown in Figure 4.11. Cysteines also react effectively... 

A comparison of the structures of penicillin and Dalanyl-Dalanine (cf. structures 41 and 42) shows that there is a great deal of similarity between the two molecules. Penicillin is essentially an acylated cyclic dipeptide of Dcysteine and Dvaline (84). As such, it contains a peptide bond, that of the /3-lactam ring, that can acylate the enzyme. Labeling studies of the peptidoglycan transpeptidase of Bacillus subtilis indicate that radioactive penicillin reacts with a sulfhydryl group of a cysteine residue of the enzyme (86). 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

More recently attempts to generate highly selective quiescent affinity labels have been made for a number of protease and kinase targets. As examples, inhibitors of the Rhinovirus 3C protease (Mathews et al 1999) and of the epidermal growth factor receptors (Boschelli, 2002), both incorporating Michael acceptors to covalently inactivate cysteine residues in their target enzymes (Lowry and Richardson, 1981 Figure 8.6), have entered human clinical trials for the treatment of rhinovirus infection and cancer, respectively. 

Figure 3.3. Structure of the ICAT reagent. The reagent contains a biotin affinity tag that is used to isolate ICAT-labeled peptides. The reagent also contains a linker that exists in a heavy (where X= deuterium) or light form (X= hydrogen) and a reactive group with specificity towards the thiol groups of cysteine residues. Figure adapted from Gygi et al. (1999).

Figure 17.29 An expressed protein containing a mutant intein segment can undergo self cleavage to form an N-terminal cysteine residue, which then can be reacted with a thioester probe to label specifically the protein via an amide bond.

The isotope-coded affinity tag approach utilizes chemical labeling that allows quantitation when combined with mass spectrometry. ICAT is desirable because the chemical labeling takes advantage of the mass defects of monoisotopic stable isotopes. ICAT uses an ICAT reagent to differentially label protein samples on their cysteine residues. ICAT is advantageous because it permits the evaluation of low-abundance proteins and proteins at both extremes of molecular weights and isoelectric points.60... 

Affinity Labeling of Catalytic ATP Sites. Residues involved in ATP binding are potentially revealed by the use of affinity labels that are based on ATP s structure. Perhaps the most systematically studied of these compounds is 5 -fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been reported to label at least six kinases (32-A1). In the case of rabbit muscle pyruvate kinase such work has Indicated the presence of a tyrosine residue within the metal nucleotide binding site and an essential cysteine residue located at or near the free metal binding site (32). A similar reagent, 5 -FSBGuanosine, revealed the presence of two cysteine residues at the catalytic site of this same enzyme, both distinct residues from those modified by 5 -FSBA (33,34). With yeast pyruvate kinase both tyrosine and cysteine residues were modified by 5 -FSBA at the catalytic site ( ), and with porcine cAMP-dependent protein kinase a lysine residue was labeled at the active site (36). 

Fig. 24. Methanethiosulfonate (MTS) reagent reacting with a thiol (sulfhy-dryl), typically of a cysteine residue in a protein, forming a disulfide bond and thus introducing the dye moiety (label)—SR.

Roche-Applied-Science. Protein labelling kits. 2002 Biochem. Catalog 2002, 322-323. Javitch, J. A. Li, X. Kaback, J. Karlin, A. A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice. Proc. Natl. Acad. Sci. USA 1994, 91,10355-10359. 

N-terminals of the /3 subunits. At the other end, a cysteine residue was introduced into the exposed tip of the y subunit, which was coupled to biotin, and then attached to a fluorescently labeled actin filament via a streptavidin linker. The ATP-dependent anticlockwise rotation of the 1- to 3-p.m-long actin filaments was seen in a fluorescence microscope.94 Smaller probes show that the rotation is consistent with the turnover of ATP by the FrATPase, which is consistent with a three-step motor.95,96 This implies that ATP synthesis requires that the y subunit be cranked in a clockwise direction by a rotary motor in F,. 

The diazomethyl ketone functional group was first observed to be an affinity label by Buchanan and co-workers who showed that the antibiotic azaserine, an O-diazoacetyl derivative, 9 inhibited an enzyme in the biosynthesis of purine by alkylation of a cysteine residue. 10 The acid protease pepsin was then observed to be inhibited by peptidyl diazomethyl ketones in the presence of copper ions with the resulting esterification of an aspartate residue. 11 Two peptidyl diazomethyl ketones, Z-Phe-CHN2 and Z-Phe-Phe-CHN2, were found to irreversibly inactivate papain, a cysteine protease. 12 Since these reports, many peptidyl diazomethyl ketones have been prepared primarily as inhibitors of various cysteine proteases. 7 Peptidyl diazomethyl ketones are also synthetic intermediates and have been used to prepare chloromethyl ketones (Section 15.1.3), 13 bromomethyl ketones (Section 15.1.3), acyloxymethyl ketones, 14 and (i-peptides. 15 A few peptidyl diazoalkyl ketones have been reported. 16,17 ... 

Creatine kinase is a dimer (molecular weight 82 000), with an active site on each monomer. The activating metal binds to the ATP only, i.e. type I. Spin labelling of the cysteine residue at the active site has allowed distance measurements to Mnu. It appears that Mn remains bound to the a- and /3-phosphoryl groups in ADP. 

To gain quantitative data without the inherent problems associated with gel electrophoresis would seem to be an added advantage. ICAT, (Fig. 17.10), also based on LC, labels reduced cysteine residues from two sample states with a multipart tag. This tag consists of an affinity tag to bind to AC media along with an isotopically coded linker, generally heavy and light forms... 

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