Chemistry Reference
In-Depth Information
GPI-anchored
pro
teins
Transmembrane
proteins
Src-family
kinase
proteins
FIGURE 3.4.
PAL strategies for cross-linking within the core of a membrane using agents
such as
34
, and on the outer surface of the membrane using agents such as
35
.
polar environment ideally suited for nitrenium ion formation. This complex ensem-
ble consists of the aforementioned cardiolipin units and at least eight protein
subunits. In spite of the apparent mismatch of PAL agents with environments,
both agents afforded significant cross-linking to different protein subunits with
varying efficiencies, but in general, the PAL agents on the nonpolar tail of cardiolipin
were more efficient cross-linking agents than the PAL agents on the polar head
group. While the information gleaned from this type of experiment provides useful
correlations about the proximity of the various subunits, the cross-linking processes
are derived from a variety of very different reactive intermediates, most of which
require microseconds or longer to establish the critical cross-linking bond, and thus,
may wander from optimal binding sites. If PAL strategies are to be advanced to the
next level of identifying specific amino acid residues and other biological constitu-
ents involved in the optimal bonding sites, then the PAL agents will have to be
selected with specific target environments and amino acid residues in mind.
Sex hormones,
,
104
and vitamin D
105-109
receptors have also been explored
using 4-azido-2-nitrophenyl amine PAL agents (Scheme 3.18). Two strategies have
been used in efforts to identify the binding sites of vitamin D
3
. One of these has been
to isotopically label either the Vitamin D
3
moeity
106
or the PAL moiety
107
with
tritium,
36
, respectively, and look for radioactive proteins produced by
irradiation of the protein-vitamin D
3
mixture. In the second and more recent study,
the
14
C-double labeling reagent,
37a
and
37b
38
, was used in which the proteins first became
