Chemistry Reference
In-Depth Information
(a)
O
O
P
O
Protein
O
Cell extract
incubation
O
P
O
Purification /
Detection
O
N
3
Labeled
lipidated
protein
N
3
Tagged
lipid substrate
(b)
O
O
O
N
3
R
O P
O P
O
SCoA
O
O
42a-b
39
a
:R=N
3
N
3
b
:R=
OH
40
O
O
R
R
H
OH
OH
43a-b
41a-b
a
:R=N
3
a
:R=(CH
2
)
2
N
3
b
:R=
b
:R=
FIGURE 4.10
Investigation of covalent protein lipidation using bioorthogonal labeling. (a)
Cartoon depicting the covalent labeling of target using azide-tagged lipid substrate analogs.
(b) Examples of structures used to infiltrate covalent protein modification using tagged lipid
substrate derivatives. (See color plate section.)
of
S-
farnesyl or
S
-geranylgeranyl moieties onto cysteine. The importance of covalent
protein lipidation is represented by the role it plays in regulating proteins in the Ras
superfamily, which undergo a switching mechanism controlled by the identities of
added lipid chains [84]. When Ras is only farnesylated, it is localized at the endo-
plasmic reticulum (ER) membrane. Upon the introduction of one or more palmitoyl
groups, cycling to the inner leaflet of the plasma membrane is triggered, where Ras
becomes activated for its pivotal role in promoting cell growth. Due to the signifi-
cance of protein lipidation, numerous recent studies have exploited the incorporation
of lipid moieties bearing azide or alkyne onto proteins to study covalent lipid addition
(Fig. 4.10a). Due to the large volume of work in this area, and the fact that these efforts
have been the subject of recent reviews [85-87], we will only briefly summarize this
work herein.
In an early example of using bioorthogonal reporters to characterize protein lipida-
tion, Zhao and coworkers reported studies employing azido-farnesyl pyrophosphate
(
39
) as a substrate analog for farnesyltransferases [88]. Initially,
39
was shown to
be an effective substrate in vitro using the RC peptide as a representative sequence
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