Biology Reference
In-Depth Information
creating recombinant binding sites for antitumor drugs, obtaining well-defined sub-
strates for the enzymes responsible for tubulin posttranslational modification, or bac-
terial microtubule-like polymeric trails for motor proteins. Several divergent
sequences from the surface loops of bacterial tubulin have already been replaced
by the corresponding eukaryotic sequences, yielding soluble folded chimeras. We
describe the purification protocol of untagged bacterial tubulin BtubA/B by means
of ion exchange, size exclusion chromatography, and an assembly-disassembly cy-
cle. This is followed by methods and examples to characterize its assembly, employ-
ing light scattering, sedimentation, and electron microscopy.
INTRODUCTION AND RATIONALE
Tubulin-like proteins share the structural fold of eukaryotic tubulin and are wide-
spread in the prokaryotic world. These include essential cell division protein FtsZ
(widely distributed through bacteria and archaea; Erickson, Anderson, & Osawa,
2010 ), bacterial tubulin BtubA/B (found only in several Prosthecobacter species;
Jenkins et al., 2002 ), and the plasmid partition protein TubZ (present in large toxin
encoding plasmids from Bacillus species; Larsen et al., 2007 ). A TubZ has also been
found in a partition system encoded by phage cs-t of Clostridium botulinum ( Oliva,
Martin-Galiano, Sakaguchi, & Andreu, 2012 ), and a related protein PhuZ is encoded
by phage 201phi2.1 of Pseudomonas chlororaphis ( Kraemer et al., 2012 ).
Bacterial tubulin is the closest known prokaryotic homolog of eukaryotic tubulin.
The btubA and btubB genes were discovered during the genome sequencing of the
bacterium Prosthecobacter dejongeii ( Jenkins et al., 2002 ). They perform an unclear
cytoskeletal function in several Prosthecobacter species, whose genomes also en-
code the ftsZ essential for bacterial cell division ( Pilhofer, Rosati, Ludwig,
Schleifer, & Petroni, 2007 ). BtubA/B share higher sequence identity with eukaryotic
tubulin (
35%) than the other tubulin structural homologs FtsZ (
17%), TubZ and
PhuZ (
10%). Both proteins show a surprisingly high structural similarity with an
root-mean square deviation (RMSD) of only of 1.3-1.5 ˚ with
<
-tubulin,
whereas when compared with FtsZ from Methanococcus jannaschii , the RMSD is
2.7 ˚ ( Schlieper, Oliva, Andreu, & Lowe, 2005 ). A structural alignment between
the bacterial and eukaryotic tubulin sequences is shown in Fig. 17.1 . Bacterial tubu-
lin includes the typical C-terminal helices H11 and H12 present in eukaryotic tubulin,
which is the more divergent region between tubulin-like proteins. Though BtubA and
BtubB lack the highly acidic 15-20 residue tails of eukaryotic tubulin that participate
in interactions with microtubule-associated proteins and motors ( Nogales, 2000 ),
only BtubA has a charged C-terminal extension. Bacterial tubulin is homogeneous,
lacking the isotype diversity and the posttranslational modifications of eukaryotic
ab
-or
a
b
-tubulin. The ease of expression and better stability of bacterial tubulin compared
to eukaryotic tubulin make it an attractive framework for tubulin engineering, in
addition to yeast tubulin ( Ayaz, Ye, Huddleston, Brautigam, & Rice, 2012 ).
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