Protein Engineering

Proteins are the most abundant biomacromolecules in cells, constituting up to 50% of the dry weight of cells. In eukaryotes, proteins are produced in the ribosome, where a messenger RNA (mRNA) carries the code for the primary sequence of the protein, and is read by aminoacylated transfer RNA (aa-tRNA). The code, the genetic code, contains 64 triplet codons, of which 61 codes are for 20 different amino acids, which we call cognate, canonical, or proteinogenic amino acids—that is building blocks for protein biosynthesis. The last three codons, UAG (amber), UAA (ochre), and UGA (opal), are stop codons, also known as nonsense codons. Thus, eukaryotic proteins are generally made up of the 20 proteinogenic amino acids, although in recent years two extra amino acids have been added to this repertoire. The 21st amino acid is selenocysteine, which is found in prokaryotes and eukaryotes and where the sulfur of cysteine is replaced by selenium and the 22nd amino acid is pyrrolysine, where the e-amino group of lysine is derivatized with b-methylpyrroline (Figure 4.6).

Methods for the residue-specific incorporation of close analogs of natural amino acids have existed for many years, where the depletion of one amino acid and the addition of another, structurally related unnatural amino acid, allows the incorporation of this amino acid. A typical example is the incorporation of selenomethionine (Se-Met), in place of methionine, which is used in structural studies of proteins, as the heavy atom selenium may help in solving the phase problem in x-ray crystallography. Here, we will focus on approaches for the site-specific incorporation of unnatural amino acids into

Selenocysteine

Pyrrolysine

FIGURE 4.6 The 21st and 22nd amino acids, selenocysteine and pyrrolysine, respectively, which are obtained by the conversion of serine and lysine.

Selenocysteine

Pyrrolysine

FIGURE 4.6 The 21st and 22nd amino acids, selenocysteine and pyrrolysine, respectively, which are obtained by the conversion of serine and lysine.

proteins; specifically, strategies allowing the site-specific incorporation of unnatural amino acids using the cells own protein synthesis machinery as well as semisynthetic techniques will be discussed.

In general, the use of chemical, rather than conventional genetic methods, to alter protein structure and function offers exciting possibilities. Genetic methods are generally limited to the use of the 20 proteinogenic amino acids, which contain a finite number of functional groups. Nature has increased the diversity by a large number of PTMs (Figure 4.7), which are normally not attainable by genetic methods. Thus, by combining the principles and tools of chemistry with the synthetic strategies and processes of living organisms, it is possible to generate proteins with novel functions. Such proteins can be applied in structural and functional studies of proteins, in ways previously considered unattainable.

The possibilities for generating novel proteins are endless. As previously mentioned, the incorporation of PTMs is a key feature, which allows addressing biological importance of such modifications in great detail. PTMs that can be mimicked are group additions, such as phosphorylation, glycosylation, and lipidation, and the modification of parent amino acids also includes methylation, acetylation, and hydroxylation (Figure 4.7). Another class of modification is those that incorporate biophysical probes or reactive handles, for further derivatization, examples include site-specific labeling with 13C- or 15N-labeled amino acids for biological NMR studies, incorporation of fluorescent amino acids or amino acids containing photolabile groups such as benzophenone (Figure 4.7). Amino acids with reactive groups for selective derivatization are also of great interest; such groups could be azides or alkyne groups (Figure 4.7) to be used in the Huisgen 1,3-dipolar cycloaddition to furnish 1,2,3-triazoles, also known as "click chemistry." Another example is the introduction of ketone functionalities that can be selectively modified, for example, with polyethylene glycol (PEG) linkers. Finally, very subtle changes of proteins, such as the incorporation of D-amino acids, close analogs of encoded amino acids (Figure 4.7), and the modification of the amide backbone can also

Benzophenone (c) derivative

Alkyne derivative of tyrosine

FIGURE 4.7 Modification and incorporation of amino acids that can be achieved by applying chemically based methods. (a) PTMs, such as hydroxylation and phosphorylation. (b) Close analogs of encoded amino acids, arginine, where the subtle modification of the guanidine group is included. (c) Biophysical probes, such as benzophonene, which is a photolabile group and an alkyne derivative that can be used in "click chemistry."

Benzophenone (c) derivative

Alkyne derivative of tyrosine

FIGURE 4.7 Modification and incorporation of amino acids that can be achieved by applying chemically based methods. (a) PTMs, such as hydroxylation and phosphorylation. (b) Close analogs of encoded amino acids, arginine, where the subtle modification of the guanidine group is included. (c) Biophysical probes, such as benzophonene, which is a photolabile group and an alkyne derivative that can be used in "click chemistry."

be introduced. This allows very fine-tuned studies of, for example, ligand-receptor interactions and protein function in general and has been described as "protein medicinal chemistry."

A number of technologies have been developed to achieve this objective and it is now possible to generate proteins containing, in principle, any functionality. In the following sections, we will focus on two general methods that allow this: (1) unnatural mutagenesis, which allows the site-specific incorporation of unnatural amino acids into protein and (2) ligation-based strategies, which allows semisynthesis of proteins and thereby the incorporation of a wide range of unnatural functionalities into proteins.

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