Biomedical Engineering Reference
In-Depth Information
proteins; specii cally, strategies allowing the site-specii c 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 struc-
ture and function offers exciting possibilities. Genetic methods are generally limited to the use of
the 20 proteinogenic amino acids, which contain a i nite 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 incor-
poration of PTMs is a key feature, which allows addressing biological importance of such modii -
cations in great detail. PTMs that can be mimicked are group additions, such as phosphorylation,
glycosylation, and lipidation, and the modii cation of parent amino acids also includes methylation,
acetylation, and hydroxylation (Figure 4.7). Another class of modii cation is those that incorporate
biophysical probes or reactive handles, for further derivatization, examples include site-specii c
labeling with
13
C- or
15
N-labeled amino acids for biological NMR studies, incorporation of l uores-
cent 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 modii ed, 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 modii cation of the amide backbone can also
HO
O
2
H
2
O
N
N
O
O
Proline
4-Hydroxy-proline
HN
NH
2
O
NH
2
O
O
-
NH
NH
NH
O
-
OP
AT P
ADP
OH
O
H
H
H
H
H
O
O
O
O
O
Serine
O
-Phosphate-serine
Arginine
Citrulline
N
-Acetyl-ornithine
(a)
(b)
O
O
H
H
O
O
Benzophenone
derivative
Alkyne derivative
of tyrosine
(c)
FIGURE 4.7
Modii cation 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 modii cation 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.”
