Biomedical Engineering Reference
In-Depth Information
thus synthesis is easier and also the stability of PNA is increased compared to DNA. Another promi-
nent example of DNA, or rather RNA, modii cation is LNA, where the ribose moiety of an LNA
nucleotide is modii ed with an extra bridge connecting the 2
′
and 4
′
carbons (Figure 4.5). The
bridge locks the ribose in the 3
-endo structural conformation, which is supposed to be an important
bioactive conformation. This locked conformation of LNA enhances base stacking and backbone
preorganization, as well as increases the stability of the nucleic acids. Both PNA and LNA are being
pursued commercially as potential drug candidates, as well as diagnostic tools. Polysaccharides,
such as starch and glycogen, are highly important biomacromolecules, and the term “glycomics”
has been introduced analogous to genomics and proteomics, to describe the comprehensive study
of sugars in organisms, and glycomics is a subset of studies of sugars in biology in general, termed
glycobiology. Carbohydrates also play vital roles as posttranslational modii cations (PTMs) of gly-
coproteins and in the remainder of this chapter focus will be on proteins and methods for modifying
proteins.
′
4.3.1 P
ROTEIN
E
NGINEERING
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 dif-
ferent amino acids, which we call cognate, canonical, or proteinogenic amino acids—that is build-
ing 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-specii c 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 incor-
poration 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-specii c incorporation of unnatural amino acids into
N
CH
3
O
NH
Se
OH
OH
H
2
N
H
2
N
O
O
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.
