Biology Reference
In-Depth Information
causes the MTs and any bound MAPs to separate out of solution (the supernatant) and
form a pellet. The supernatant and pellet fractions are then assayed to determine the
protein concentration in each fraction by SDS-PAGE and densitometry analysis. The
cosedimentation method relies on the assumption (verified by appropriate controls)
that the MTs will form a pellet regardless of any MAPs that may be bound and that
the MAPs will not spin down in the absence of binding to MTs. While the simulated
curves inMTBindingSimwere designed with cosedimentation assays inmind, the data
will be the same for any method of investigating MAP-MT binding, such as fluores-
cence anisotropy, SPR, or tryptophan fluorescence. MTBindingSim follows the stan-
dard convention of writing the concentration of MTs, [MT], to mean the concentration
of polymerized tubulin dimers (the “MT polymer”). MAP-MT-binding experiments
are usually performed in the presence of MT stabilizers such as taxol, so the concen-
tration of unpolymerized tubulin dimers will be minimal and can be neglected. Exper-
iments performed with a high concentration of unpolymerized tubulin dimers will
present additional complications in interpreting binding curves and cannot (currently)
be modeled with MTBindingSim.
Vary [MT]
: In this assay, the concentration of A (a generic MAP) is held constant
and the concentration of MT is varied. This is the “standard” binding experiment.
The fraction of A bound to MT is graphed on the
y
-axis. The user can choose either
the total MT concentration or the free MT concentration to be graphed on the
x
-axis.
Note that when the
x
-axis is set to [MT] free, this experiment shows the familiar
Langmuir isotherm. The quick method of determining
K
D
from the concentration
of MT where 50% of A is bound
only
works when the
x
-axis plots [MT] free, not
when it plots [MT] total.
Vary [A]
: In this assay, the concentration of MT is held constant and the concen-
tration of A is varied. There are three possible ways of graphing data from this kind of
simulation. The first is a Scatchard plot where the
x
-axis is [A] bound and the
y
-axis
is [A] bound/[A] free. In this kind of plot, binding data will be linear for a simple 1:1
interaction, and a deviation from linearity indicates cooperativity or other types of
nonsimple binding. The other two plots have [A] bound on the
y
-axis and the
x
-axis
set to either [A] free or [A] total.
Competition
: In this assay, there are two MT-binding proteins, A and B. The con-
centration of A and MT is held constant and the concentration of B is varied. The
y
-axis is the fraction of A bound and the
x
-axis is the total concentration of B. In
the present version of MTBindingSim, this kind of experiment can only be simulated
when both A and B bind to MTs with simple 1:1 binding.
23.3
BINDING MODELS IN MTBindingSim
The binding models simulated in MTBindingSim were chosen to reflect commonly
seen or hypothesized mechanisms of MAP-MT binding. While MTBindingSim was
written specifically with MAP-MT binding in mind, most of its models have broader
applicability. Only the “seam and lattice binding” model is MT specific as it relies on
the unique structure of the MT. The “MAPs dimerize” and “pseudocooperativity”
