Protein lysine residues

The immobilization strategies are of particular interest. The authors reasoned that the use of aldehydes to tether proteins to the solid phase could be ideal for certain protein-protein interaction studies. Since many protein-lysine residues are available for coupling to aldehydes via Schiff s base, a number of spatial orientations are possible. Such random oriented attachments would permit exposure of various surfaces of a protein to the solution, and new protein-protein interactions would be potentially possible. 

It is probably the f-amino groups of the proteins lysine residues, that react with glutaraldehyde forming a simple Schiffs base as suggested by Brarmer-Jorgensen (1978). 

Trinitrobenzene sulfonic acid (TNBS) is known to react with protein lysine residues, forming colored trinitrophenylated (TNP) derivatives with absorption maxima at 345 mp, (46). When E. coli pyrophosphatase was incubated with a 400-fold molar excess of the reagent, there was progressive decay of enzymic activity accompanied by increasing absorbance of the solution at 345 mp. (Fig. 5). Half the enzymic activity had disappeared by the time 16% of the protein lysines had been modified eventually (6 hr) all 98 enzyme lysines reacted (Table III and Fig. 5). Isolation and hydrolysis of the product showed total conversion of all lysine to e-TNP-lysine without formation of any other... 

Figure 23.2. Ubiquitin. The structure of ubiquitin reveals an extended carboxyl terminus that is activated and linked to other proteins. Lysine residues also are shown, including lysine 48, the major site for linking additional ubiquitin molecules.

Fig. 33. Synthesis of an N-hydroxysuccinimidyl derivative of his-adamantyl dioxetane, suitable tor covalently labeling the amino groups of hapten derivatives or protein lysine residues. Adapted from Hummelen et al. (H19), with permission.

In the 1970s, O Leary and Samberg described the reaction of 2,4,6-trimethyl-pyrylium perchlorate 57 with a-chymotrypsin and observed the formation of a protein-pyridinium conjugate [94], Later on, the reaction of the tri-substituted pyrylium salt 58 with the proteins gelatin and a-chymotrypsin [95], glycophorin [96,97] and the Na /glucose cotransporter protein [98] was reported. In all cases, the reaction happened to exclusively involve the proteins lysine residues and formation of the corresponding pyridinium adducts were observed. 

Many pharmaceutical compounds are weak acids or bases that can be analyzed by an aqueous or nonaqueous acid-base titration examples include salicylic acid, phenobarbital, caffeine, and sulfanilamide. Amino acids and proteins can be analyzed in glacial acetic acid, using HCIO4 as the titrant. For example, a procedure for determining the amount of nutritionally available protein has been developed that is based on an acid-base titration of lysine residues. ... 

FIGURE 10.23 The folding of halorhodopsin with the transmembrane segments indicated. The only lysine residue in the protein is Lys , to which the retinal chromophore is covalently linked. 

FIGURE 18.32 Biotin is covalently linked to a protein via the e-amino group of a lysine residue. The biotin ring is thus tethered to the protein by a 10-atom chain. It functions by carrying carboxyl groups between distant sites on biotin-dependent enzymes. 

Histone acetylation is a reversible and covalent modification of histone proteins introduced at the e-amino groups of lysine residues. Histones and DNA form a complex - chromatin - which condenses DNA and controls gene activity. Current models interpret histone acetylation as a means to regulate chromatin activity. 

Small tfbiquitin-like modifier represents a family of evolutionary conserved proteins that are distantly related in amino-acid sequence to ubiquitin, but share the same structural folding with ubiquitin proteins. SUMO proteins are covalently conjugated to protein substrates by an isopeptide bond through their carboxyl termini. SUMO addition to lysine residues of target proteins, termed SUMOylation, mediates post-transla-tional modification and requires a set of enzymes that are distinct from those that act on ubiquitin. SUMOylation regulates the activity of a variety of tar get proteins including transcription factors. 

Small Ubiquitin-like modifier (SUMO) is a conserved protein that is ubiquitously expressed in eukaryotes and is essential for viability. It serves as a reversible posttranslational modifier by forming an isopeptide bond with lysine residues in many target proteins, in a catalytic process termed SUMOylation. SUMOylation of proteins results in altered inter- or intramolecular interactions of the modified target (Fig. 1). 

Ubiquitin/Proteasome. Figure 2 Functional consequences of ubiquitin linkage. Substrates (blue bars) are linked via lysine residues (K) to ubiquitin or ubiquitin chains, (a) Attachment of chains connected via Lysines in position 48 of ubiquitin (K48) targets substrates for proteasomal degradation. In contrast modification of one (b) or multiple (c) lysines by a single ubiquitin molecule mediates novel protein interactions or initiates endocytosis. Conjugation of K63-linked polyubiquitin (d) alters protein function and can also serve as a signal for endocytosis. 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

Fig. 15 Amino acid sequences of artificial extracellular matrix (aECM) proteins. Each protein contains a TV tag, a histidine tag, a cleavage site, and elastin-like domains with lysine residues for crosslinking. The RGD cell-binding domain is found in aECM 1, whereas aECM 3 contains the CS5 cell-binding domain. aECM 2 and aECM 4 are the negative controls with scrambled binding domains for aECM 1 and aECM 3, respectively. Reprinted from [121] with permission from American Chemical Society, copyright 2004...

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