Biology Reference
In-Depth Information
isolated BtubA contained 0.13
0.06 GTP and 0.53
0.11 GDP and isolated BtubB
contained 0.08
0.09 GDP. Protein concentration can be deter-
mined spectrophotometrically in 6 M guanidinium chloride (as above), employing
the extinction coefficients BtubA 47,700 M
1
cm
1
and BtubB 38,780 M
1
cm
1
at 280 nm, after subtracting the contribution of bound guanine nucleotide (as de-
scribed in purification section 5 above). Separated untagged BtubA and BtubB were
not able to assemble by themselves (in homo-polymers), and titration experiments
showed that the maximum level of heteropolymer recovery by centrifugation is when
BtubA and BtubB are mixed in an equimolar ratio.
0.07 GTP and 0.47
17.2.3
BtubA/B chimera containing eukaryotic sequences
In a previous work, we constructed bacterial-eukaryotic tubulin chimeras and ana-
lyzed their biochemical behavior (
Martin-Galiano et al., 2011
). These chimeras, con-
taining eukaryotic loop sequences 1, M, and S (see
Section 1
) from
-tubulin,
were purified as described above, as well as the wild-type protein for comparison
purposes. We showed that every construct was folded and shared similar average
secondary structure compared with wild-type BtubA/B when analyzed by circular
dichroism. Furthermore,
-or
a
b
these chimeras assembled into polymers similar
to
the wild-type protein. This included introducing the M loop of
-tubulin into BtubA
a
and that of
-tubulin into BtubB, or vice versa, the M loop of
-tubulin into
b
a
BtubB and that of
-tubulin into BtubA. However, upon successive introduction
of the eukaryotic sequences, the overall expression and purification yield decreased.
A special case were the chimeras containing the
b
-tubulin S-loop, because only one-
third of the expressed protein was soluble and only a small proportion of that was
able to assemble during the polymerization step (section 4 in purification protocol)
(
Fig. 17.3
). Therefore, when working with BtubA/B constructs with suspected inhib-
ited polymerization activity, this step should be omitted.
a
17.2.4
Methods to study assembly of purified BtubA/B
There are several methods for determining the ability of BtubA/B and their constructs
to polymerize into large head-to-tail ordered structures. The more frequent methods
include right-angle light scattering, sedimentation, and electron microscopy.
The time course of BtubA/B assembly can be conveniently monitored by right-
angle light scattering in a fluorometer with excitation and emission wavelengths set
at 350 nm, employing a temperature controller. As other tubulin-like proteins,
BtubA/B assembly requirements include Mg
2
þ
,K
þ
, and a guanosine nucleotide tri-
phosphate. It is important to consider that untagged BtubA/B hardly polymerizes in
typical microtubule assembly buffers. Instead, we use the polymerization buffer
(20 mM Tris, 500 mM KCl, 1 mM EGTA, pH 7.5) and add 5 mM MgCl
2
followed
by GTP (0.1 or 1 mM), or the slowly hydrolysable analogue GMPCPP (0.1 mM), to
start the assembly reaction at 25
C(
Martin-Galiano et al., 2011
). To ensure the
