Biomedical Engineering Reference
In-Depth Information
Fig. 9.4
Binding curve of
His
6
-tagged Hsp70 to a
fl uorescein-conjugated RNA
substrate encoding the ARE
from tumor necrosis factor a
(TNFa) mRNA (
solid
circles
). The best- fi t function
is consistent with a 1:1
binding interaction with
K
= 4.0 × 10
7
M
−1
(
K
d
= 25 nM). A residual runs
test (
bottom
) shows that this
binding model is consistent
with the observed anisotropy
data across the entire protein
titration. A separate protein
titration experiment shows no
significant Hsp70 binding to
a similarly sized fragment of
b-globin mRNA (
open
circles
)
useful for identifying the minimum number of RNA-containing complexes formed
during protein binding reactions, which in turn yields a valuable starting point for
defining binding models that will then be quantitatively characterized by anisot-
ropy-based assays.
In Fig.
9.4
and all other binding curves presented in this chapter, the protein
concentration has been plotted on a logarithmic scale instead of a linear scale. When
anisotropy isotherms are presented in the literature, however, some investigators
will opt to present the
x
-axis on a linear scale. This method of presentation is com-
pletely accurate, but complicates facile assessments of binding mode and makes
assessment of deviations between data and the model across the range of tested
protein concentrations more difficult.
For reactions best described by two rounds of protein binding to a common RNA
substrate, the equation becomes
AAKPAKKP
+
[]
+
[]
2
A
=
R
PR
1
P
2
R
1
2
(9.17)
t
2
1[
+
KP KK P
]
+
[
]
1
1
2
Binding of the human p37
AUF1
protein to an RNA substrate follows this two-step
binding model as shown in Fig.
9.5
. AUF1 is a family of four dimeric proteins gen-
erated by alternative splicing of a common pre-mRNA (Wagner et al.
1998
) that has
diverse functions in control of mRNA decay, translation, and telomere maintenance
(Lu et al.
2006
; Liao et al.
2007
; Sarkar et al.
2011
; Eversole and Maizels
2000
) .
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