Biology Reference
In-Depth Information
11.1.7.4 Chromatography on Hydroxylapatite .................................................. 166
11.1.7.5 Size Exclusion Chromatography......................................................... 167
11.2 Discussion......................................................................................................167
References ............................................................................................................. 169
Abstract
The tubulin heterodimer consists of one
-tubulin polypeptide. Neither pro-
tein can partition to the native state or assemble into polymerization competent hetero-
dimers without the concerted action of a series of chaperone proteins including five
tubulin-specific chaperones (TBCs) termed TBCA-TBCE. TBCA and TBCB bind to
and stabilize newly synthesized quasi-native
- and one
a
b
-tubulin polypeptides, respec-
tively, following their generation via multiple rounds of ATP-dependent interaction
with the cytosolic chaperonin. There is free exchange of
b
-and
a
b
-tubulin between TBCA
and TBCD, and of
a
-tubulin between TBCB and TBCE, resulting in the formation of
TBCD/
b
andTBCE/
a
, respectively. The latter two complexes interact, forming a super-
complex (TBCE/
heterodimer occurs via inter-
action of the supercomplex with TBCC, which results in the triggering of TBC-bound
b
-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the
supercomplex and the release of E-site GDP-bound heterodimer, which becomes poly-
merization competent following spontaneous exchange withGTP. The tubulin-specific
chaperones thus function together as a tubulin assembly machine, marrying the
/TBCD/
). Discharge of the native
/
a
b
a
b
-and
a
-tubulin subunits into a tightly associated heterodimer. The existence of this evolution-
arily conserved pathway explains why it has never proved possible to isolate
b
-or
a
-tubulin as stable independent entities in the absence of their cognate partners, and
implies that each exists and is maintained in the heterodimer in a nonminimal energy
state. Here, we describe methods for the purification of recombinant TBCs as biolog-
ically active proteins following their expression in a variety of host/vector systems.
b
INTRODUCTION
The
-tubulin heterodimer was originally thought to assemble spontaneously via
association of the two constituent polypeptides, with a binding constant in the micro-
molar range (
Detrich & Williams, 1978
). More recent measurements based on plas-
mon resonance suggest a dissociation constant in the range of 10
11
M(
Caplow&Fee,
2002
). In any event, it has never proved possible to purify
/
a
b
a
-or
b
-tubulin in native form
-tubulin in
Escherichia coli
,
either alone or together, leads to their deposition within the host cells as completely
insoluble inclusion bodies. Attempts to recover native tubulin from these insoluble
materials have consistently proved futile, in spite of thewell-established principle that
all the information required for a given protein to assume its correct three-dimensional
structure is contained within its primary structure (
Anfinsen, 1973
). However,
in vitro
free from its counterpart. Moreover, expression of
a
-or
b
