Biology Reference
In-Depth Information
Abstract
Fluorescent tubulin can be prepared in which a fluorophore is covalently bound to the
protein at only the carboxy terminus of the
-tubulin dimer. This
two-step procedure consists of an enzymatic reaction followed by a bioorthogonal
chemical reaction. In the first step of the process, the enzyme tubulin tyrosine ligase
is used to attach a reactive tyrosine derivative, 3-formyltyrosine, to the protein. In the
second step of the procedure, a fluorophore possessing a complementary reactive
functional group, such as a hydrazine, hydrazide, or hydroxylamine, is allowed to
react with the protein under conditions that are compatible with native tubulin.
Polymerization-competent, fluorescently labeled tubulin can be prepared in just a
few hours using this protocol. The method described here should be useful for attach-
ing virtually any probe or material to tubulin at this site.
a
-subunit of the
ab
INTRODUCTION
Attaching an exogenous reporter molecule to tubulin is normally accomplished with
a reactive probe that targets a naturally occurring amino acid such as lysine. Many
lysine-reactive probes are commercially available, and protocols for performing the
labeling reactions can be found in the literature (
Hyman, et al., 1991; Peloquin,
Komarova, & Borisy, 2005; Wadsworth & Salmon, 1986
). The conditions necessary
for efficient reaction between the probe and the protein (e.g., high pH) sharply de-
crease the ability of tubulin to assemble into microtubules. It is necessary to perform
a cycle of assembly-disassembly to obtain polymerization-competent protein.
Although active, fluorescent tubulin is accessible by this method, the overall yield
of labeled protein is low, and the position of the labels and the degree of labeling
cannot be controlled.
We have developed a method by which a single probe can be attached to just
the C-terminus of
-tubulin through a two-part procedure (
Banerjee, et al., 2010
).
In part 1, a tyrosine derivative possessing a reactive functional group is attached
to the carboxy terminus of
a
-tubulin using the enzyme tubulin tyrosine ligase
(TTL). The synthetic amino acid 3-formyltyrosine (3fY) is used as the substrate,
resulting in the retyrosinated protein that now possesses a reactive aromatic
aldehyde. In part 2, a probe containing a complementary reactive functional group
such as a hydrazine, hydrazide, or hydroxylamine is allowed to react with the
3-formyltyrosinated protein. Since the two functional groups are chosen to have
orthogonal reactivity with respect to endogenous amino acid residues, the probe is
attached to tubulin at a single location.
An important factor to consider in part 1 is that the TLL uses both the tyrosine
derivative and the carboxy terminal peptide of
a
-tubulin as substrates; therefore, the
structure of each component is significant to the ligation reaction. Consider tubulin:
purified tubulin from most sources contains a mixture of isotypes and isoforms.
Isotypes of
a
a
-tubulin that terminate in E-E-Y are subject to the tyrosination/
