Thioester-containing protein

Heme coenzymes (8) with redox functions exist in the respiratory chain (see p. 140), in photosynthesis (see p. 128), and in monooxygenases and peroxidases (see p. 24). Heme-containing proteins with redox functions are also referred to as cytochromes. In cytochromes, in contrast to hemoglobin and myoglobin, the iron changes its valence (usually between +2 and +3). There are several classes of heme (a, b, and c), which have different types of substituent - Ri to - R 3. Hemoglobin, myoglobin, and the heme enzymes contain heme b. Two types of heme a are found in cytochrome c oxidase (see p. 132), while heme c mainly occurs in cytochrome c, where it is covalently bound with cysteine residues of the protein part via thioester bonds. 

Fig. 3. (Continued) cysteine-possessing protein. The protein of interest (protein 2) is expressed as CBD-intein-protein 2 precursor and purified by the chitin beads. Temperature- or pH-induced intein cleavage results in proteins with an N-terminal cysteine residue. Finally, the EPL (dotted line) of the protein thioester and the cysteine containing protein, which can be also obtained synthetically, proceeds under NCL conditions.

Many other acyl transfer reactions are important cellular processes. Thioesters of fatty acids react with cholesterol, forming cholesteryl esters in an enzyme-catalyzed reaction (Figure 22.5). These esters are the principal form in which cholesterol is stored and transported in the body. Because cholesterol is a lipid, insoluble in the aqueous environment of the blood, it travels through the bloodstream in particles that also contain proteins and phospholipids. These particles are classified by their density. 

Ligands with free amino groups can be immobilized on Afli-Gel 201 and 202 by the EDAC method. Ligands can be attached to Afii-Gel 401 by disulfide, thioester, or thioether formation. Affi-Gel 5C has a high capacity for selectively purifying SH-containing proteins. 

As summarized in Sect. 3, the synthesis of grafted polymers requires side chain functionalities on the main chain as well as chain end functionalities on the polymers forming the arms. Besides, chain end modified polymers themselves exhibit highly interesting properties. To obtain chain end modificatimis, weU-defined chain ends are a primary requirement and can be obtained via controlled polymerizatiOTi techniques and subsequent chain end modification. For example, Sumerlin and coworkers reduced the thioester of poly(iV-isopropylacrylamide) (PNIPAM prepared by RAFT polymerization) using 1-hexylamine in the presence of tributyl-phosphine to yield a thiol-terminated polymer (Scheme 13). Subsequently, a bismaleimide was used to coimect the PNIPAM and other thiols. Thereby, small organic molecules could be used as well as other polymers or thiol-containing proteins [20, 21]. 

Analyze samples by 15 % SDS-PAGE to check loading and cleavage efficiency. Combine protein thioester containing fractions and verify identity with mass spectrometry. Include always a sample of beads, because protein thioester might precipitate that will in turn reduce yield, if not realized. 

A variety of cellular and viral proteins contain fatty acids covalently bound via ester linkages to the side chains of cysteine and sometimes to serine or threonine residues within a polypeptide chain (Figure 9.18). This type of fatty acyl chain linkage has a broader fatty acid specificity than A myristoylation. Myristate, palmitate, stearate, and oleate can all be esterified in this way, with the Cjg and Cjg chain lengths being most commonly found. Proteins anchored to membranes via fatty acyl thioesters include G-protein-coupled receptors, the surface glycoproteins of several viruses, and the transferrin receptor protein. 

Figure 17.13 Expressed proteins containing a thioester intein tag can be specifically modified using a cysteine-alkyne derivative by transthioesterification followed by an internal S - N shift.

Figure 17.14 An expressed protein containing a thioester intein tag that was subsequently modified by native chemical ligation to contain an alkyne group then can be labeled using an azido-fluorescein probe by the click chemistry reaction in the presence of Cu1+.

Native chemical ligation also can be extended to the conjugation of peptides or proteins to other molecules or surfaces. For instance, Reulen et al. (2007) prepared liposomes that contained cysteine-PEG-phospholipid derivatives and then coupled thioester-modified peptides or proteins to form a protein-liposome conjugate. Using this procedure, approximately 100 molecules of a collagen binding protein could be coupled to the cysteine-containing liposomes. 

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

Figure 17.28 EPL reactions can be used to couple a fusion protein to a surface containing a thioester derivative. After cells are grown and the fusion protein expressed, a pH and temperature shift causes intein cleavage with release of the expressed protein with an N-terminal cysteine residue. Reaction with the thioester surface results in a native chemical ligation reaction that forms an amide bond linkage with the expressed protein.

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 following protocol for EPL, including purification using a CBD fusion tag followed by native chemical ligation, is based on the methods of Muir et al. (1998), Chong et al. (1997, 1998), Evans et al. (1998), Severinov and Muir (1998), and the NEB instruction manual for the IMPACT-TWIN system. The recombinant protein is recovered from the affinity column as the thioester derivative ready for reaction with a N-terminal Cys peptide or another tag containing a Cys residue. 

The CES family of proteins is characterized by the ability to hydrolyze a wide variety of aromatic and aliphatic substrates containing ester, thioester, and amide bonds (Heymann 1980, 1982). Cauxin is a member of the CES family, and is secreted from the proximal straight tubular cells into the urine in a species-, sex-, and age-dependent manner. Therefore, we postulated that cauxin was involved in an enzymatic reaction in cat urine and the products made by the reaction should vary with species, sex, and age. Based on this hypothesis, we searched for physiological substrates and products of cauxin in cat urine and identified 2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid, also known as felinine. 

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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