Cysteine residues modification

For small molecules containing sulfhydryls or for low-molecular-weight peptides containing cysteine residues, modification may proceed without deforming agents. However, for intact proteins containing both disulfides and free sulfhydryls, a denaturant and a disulfide reducing... 

Allow the reduced protein solution to cool to room temperature and add lAA from a 0.5 M stock solution to reach 20 mM concentration in the reduced cell lysate. Incubate the reaction for 30 min at room temperature in the dark to alkylate the cysteine residues. Modification of the protein thiol groups by alkylation prevents reformation of disulfide bonds due to oxidation upon exposure to air. 

Palmitoylation is the post-translational lipid modification of cysteine-residues in a variety of proteins. 

It is not only the activity that can be altered by incorporation of noncoded amino acids. Introduction of structures possessing certain chemical functions leads to the possibility of highly regioselective modification of enzymes. For example, selective enzymatic modification of cystein residues with compounds containing azide groups has led to the preparation of enzymes that could be selectively immobilized using click chemistry methods [99]. 

Chemical modifications like alkylation with (A-ethylmaleimide (NEM) or oxidation with diamide that inhibit the phosphorylation activity of the enzyme did not seem to have any significant effect on the high affinity binding site when the enzyme was solubilized in the detergent decyl-PEG [69,41]. However, in the intact membrane these treatments reduced the affinity by a factor of 2-3. The reduction of the affinity was exclusively due to modification of the cysteine residue at position 384 in the B domain [69]. Apparently, the detergent effects the interaction between the B and C domains. 

Figure 8.5 Chemical transformation of omeprazole to the corresponding sulphenamide under add conditions and the subsequent modification of an enzyme cysteine residue by the sulphenamide.

Thiolsulfonate-containing compounds can react with thiols with release of the sulfonate end of the molecule to yield disulfide derivatives. The modification reagent 2-aminoethyl-2 -aminoethanethiolsulfonate, or AEAETS, reacts with a sulfhydryl with release taurine (2-aminoethanesulfonate) to form a 2-aminoethyl-dithiol derivative (Figure 1.94). AEAETS can be used to block cysteine residues in proteins and form derivatives containing positively charged amines. 

Methyl methanethiosulfonate (MMTS) is a small reversible blocking agent for sulfhydryl groups (Thermo Fisher, Toronto Research). It reacts with free thiols to form a dithiomethane modification with release of sulfinic acid (Figure 1.122). The sulfinic acid component decomposes into volatile products, which don t affect the disulfide formed from the MMTS reaction Alkylthiosulfonates react rapidly with thiols under mild conditions at physiological pH. The MMTS compound is a liquid at 10.6 M concentration and is conveniently added to a reaction medium by pipette. Complete thiol modifications of available cysteine residues in proteins can... 

The addition of a radioactive iodine atom to a protein molecule typically has little effect on the resultant protein activity, unless the active center is modified in the process. The size of an iodine atom is relatively small and does not result in many steric problems with large molecules. The sites of potential protein modification are tyrosine and histidine side chains. Tyrosine may be modified with a total of two iodine atoms per phenolate group, whereas histidine can incorporate one iodine. Sulfhydryl modification at cysteine residues is typically unstable. 

A derivative of Sulfo-SBED containing a thiol-reactive pyridyl disulfide group on its thermo-reactive arm has been reported for modification of bait proteins containing a cysteine residue. 

P.E. Jensen, J.D. Reid, and C.N. Hunter, Modification of cysteine residues in the Chll and ChlH subunits of magnesium chelatase results in enzyme inactivation. Biochem. J. 352, 435-441 (2000). 

Some proteins can be posttranslationally modified by the addition of prenyl groups. Prenyl groups are long-chain, unsaturated hydrocarbons that are intermediates in isoprenoid synthesis. The farnesyl group has 15 carbons, and the geranylgeranyl has 20 carbons. They are attached to a cysteine residue near the end of the protein as a thiol ether (Protein-S-R). Other proteins can have a long-chain fatty acid (C14=myristoyl, C16=palmitoyl) attached to the amino terminus as an amide. These fatty acid modifications can increase the association of proteins with the membrane. 

G protein y subunits are modified on their C-terminal cysteine residues by isoprenylation [26,27]. There is now strong evidence that this modification plays a key role in anchoring the ysubunit and its associated P subunit to the plasmalemma. The importance of this anchoring is illustrated in Figure 19-2, which shows that the ability of py subunits to direct GRKs to ligand-bound receptors depends on this membrane localization. 

In contrast, pertussis toxin catalyzes the ADP-ribosyl-ation of a specific cysteine residue in Gai) G(m and Gal [1]. Only a subunits bound to their Py subunits can undergo this modification. Pertussis-toxin-mediated ADP-ribosylation inactivates these a subunits such that they cannot exchange GTP for GDP in response to receptor activation (Fig. 19-1B). By this mechanism, pertussis toxin blocks the ability of neurotransmitters to inhibit adenylyl cyclase or to influence the gating of K+ and Ca2+ channels in target neurons. However, since G is not a substrate for pertussis toxin, the toxin may not be able to block neurotransmitter-mediated inhibition of adenylyl cyclase in all cases. The Gq and Gn 16 types of G protein a subunit are not known to undergo ADP-ribosylation. 

Multiple sulfatase deficiency (MSD). The clinical presentation of MSD includes features of MLD and the MPS, with the features that resemble MLD, such as gait disturbances, psychomotor retardation and polyneuropathy predominating. MPS-like features, such as facial coarsening, hepatosplenomegaly and joint stiffness, are present to a milder and variable extent. A reduction in the activity of all sulfatases is the key diagnostic abnormality. Von Figura and associates have demonstrated that catalytic activity of all sulfatases requires a post-translational modification in which a cysteine residue (cysteine 69 in arylsulfatase A) is oxidized to an aldehyde. It is this modification that is defective in MSD [5]. 

In normal cells, the GDP/GTP-binding proteins, after protein synthesis, move to the cell membrane to which they become hooked by a hydrophobic farnesyl group. The y-subunit is anchored in the membrane by a post-translational modification of the C-terminal CAAX sequence (C - cystein, AA - aliphatic amino acids, X - methionine). This protein is first enzymatically farnesylated by a specific farnesyltransferase, then the AAX part is cleaved by specific proteases and finally the cystein residue is converted to a methyl ester. 

By means of this method, a variety of Ras proteins with different lipidation patterns could be synthesized in multimilligram amounts. For instance, proteins were generated with the natural lipid combination, i.e. a farnesyl thioether and a palmitoyl thioester. Furthermore, analogous proteins were synthesized embodying only one lipid residue in which either the farnesyl- or the palmitoyl group was replaced by a stable hexadecyl thioether. In addition, proteins were built up containing a serine instead of a cysteine residue at the critical sites which normally are lipidated. In a further series of experiments, lipidated Ras proteins which carry a fluorescent Mant group incorporated into the farnesyl-type modification were synthesized.1251... 

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