Chemistry Reference
In-Depth Information
FIGURE 3.3.
Binding of azide PAL agents to active site of chicken skeletal myosin: (a)
Docking of PAL agent
31
in myosin based upon molecular dynamics modeling.
91
(b) Docking
of PAL agent
29
in myosin-based reorientation of the conformation of the PAL agent. Major
tryptic fragments colored orange, blue, and dark green, triphosphate unit colored pale green,
nitrenium ion colored red, Lys
681
colored yellow, and Trp
130
(rabbit myosin), or Trp
131
(chicken myosin) colored purple.
with amino acids determined by Falvey and shown in Table 3.4.
78
This same
entrapment technique using
has also been applied in the study of scallop
myosin.
89,90
In this case, the entrapment cross-linking agents were Mn
2
þ
and
vanadate, since the aforementioned Co
2
þ
procedure cause precipitation of the
protein. Here again a high yield of photolabeling was achieved, about 35%, and
the major cross-link was found to be with Arg
128
in the protein sequence Arg
127
-
Arg
128
-Leu-Pro-Ile-Tyr-Thr
133
, which is the analogous position to that of the
Trp
130
in the rabbit myosin that was specifically labeled in the previous work. In both
of these studies, the structures of the cross-linked amino acids were not determined,
and in the latter study, the Arg
128
photolabeled product was found to be unstable
under the sequencing conditions. The authors speculated that this might be due to the
cross-linking bond being a nitrogen
30
nitrogen bond between the nitrene nitrogen
and arginine guanidinium nitrogen.
89
However, it might just as well have been an
adduct analogous to those shown in Scheme 3.9, where X is the guanidine group.
Adducts of this type, triaminobenzenes, are susceptible to spontaneous air oxidation,
which if followed by hydrolysis could easily regenerate the arginine residue. Clearly
it would be most desirable to firmly establish the structures of the PAL amino acid
adducts for this widely used PAL agent. For that matter, this same problem presents
an issue with many PAL agents for which the literature is rife with kinetic, transient,
and labeling studies of products of unknown or presumed structure. In an extension
of this scallop myosin work, which was conducted in the absence of Mg
2
þ
or Ca
2
þ
ions, addition of these ions to the photolabeling experiment shifted the predominant
labeling site from Arg
128
to Cys
198
.
90
This study indicates that Ca
2
þ
plays an
