Peptide Protein Ligation

A conceptually different strategy for the modification of proteins is to employ methods based on solid-phase peptide synthesis (SPPS) for the generation of proteins. This would allow the incorporation of principally any amino acid, and thus circumvent the problems of incorporating D-amino acids, which is not feasible by unnatural mutagenesis. SPPS has in a few cases been applied for the synthesis of proteins, although yields are generally rather low. The first example was the synthesis of ribonuclease A (124 residues) by Bruce Merrifield in 1966 and since then a few other proteins have been prepared by this approach, most notably HIV protease (99 residues), which enabled structural characterization of the protein with inhibitors bound.

However, SPPS is generally limited to the preparation of up to 40-60 amino acid peptides, whereas most proteins are considerably larger. Therefore, there has been a considerable interest in developing methods that are not confined to these restrictions and in 1994, a strategy for the preparation of proteins from peptide fragments was introduced, called native chemical ligation (NCL, Figure 4.10). In NCL, two or more unprotected peptide fragments can be ligated together, generating a (native) cysteine residue in the ligation site. The ligation requires a peptide with a C-terminal protein thioester and a peptide with an N-terminal cysteine residue: the thiolate of the N-terminal cysteine attacks the C-terminal thioester to affect transthioesterification, followed by the formation of an amide bond after S ® N acyl transfer (Figure 4.10). The reaction takes place in aqueous buffer and generally proceeds in good to excellent yield.

Thus, NCL is a very useful approach for the total chemical synthesis of proteins and has been used for the preparation of numerous proteins, including glycoproteins and proteins with fluorescent labels. An example is the synthesis of an analog of erythropoietin (EPO), which was derivatized with monodisperse polymer moieties in order to improve the duration of action in vivo. The 166-residue

9 0 transthioesterification h N JL fl

S--N acyl shift

Peptide 2

Peptide 1

Peptide 1

Peptide 2

Protein

Protein

FIGURE 4.10 Principles of NCL and EPL. (a) NCL: a peptide with an N-terminal cysteine and another peptide with a C-terminal thioester can be ligated together. Initially, a reversible transthioesterification takes place and subsequently S ® N acyl shift, leading to a cysteine in the ligation site. (b) EPL is applying the same principles, but one of the reactants is a recombinantly expressed protein, which allows the semisynthesis of larger proteins.

protein was prepared by the ligation of four peptide fragments, two of which were modified with the polymer and the EPO analog displayed improved properties in vivo compared to EPO.

In 1998, an extension of the NCL principles was introduced, called expressed protein ligation (EPL). The technology applies the same reaction as in NCL, but in contrast to NCL, one of the components is a protein, rather than a peptide (Figure 4.10). The protein is expressed as the so-called intein construct, which allows the formation of a protein thioester, which subsequently can be reacted with a peptide with an N-terminal cysteine in an NCL generating a full-length protein (Figure 4.10). Thus, the EPL methodology combines the advantages of molecular biology with chemical peptide synthesis, and enables the addition of unnatural functionality to a recombinant protein framework.

EPL has been applied in studies of several proteins and here only a few noteworthy examples are provided. Histone complexes are important for the storage of DNA and have flexible N-terminal tails that are heavily modified by PTMs, and is of general importance for epigenetic gene regulation (see Chapter 23). EPL has been applied to prepare full-length, ubiquilated H2B and subsequently used to demonstrate a direct cross talk between PTMs on different histones. The list of proteins prepared by EPL was extended to include integral membrane proteins, specifically the potassium channel KcsA, which is a tetrameric assembly of identical subunits (see also Chapter 13). EPL was used to prepare KscA subunits (122 residues), which were then refolded and reconstituted into lipid membranes. In subsequent studies, unnatural mutations, such as D-alanine and amide-to-ester mutation, in the selectivity filter of the channel have revealed the important information of the function of this important segment of the potassium channel.

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