Biology Reference
In-Depth Information
detyrosination cycle by the endogenous enzymes, and these are the major isotypes
found in most tissues (
Erck, Frank, & Wehland, 2000
). Tyrosinated tubulin (Tyr-
tubulin) in this population can be effectively detyrosinated by carboxypeptidase A
(CPA) to yield “Glu-tubulin.”
In vivo
, Glu-tubulin may be further processed by an
endogenous glutamylase to form
-2 tubulin, which is not a substrate for TTL
(
Janke & Bulinski, 2011
). Fortunately, Glu-tubulin is a much poorer substrate
for CPA than Tyr-tubulin and prolonged treatment of
D
ab
-tubulin with CPA does
not produce
-2 tubulin.
A number of studies have demonstrated that TTL exhibits some flexibility in the
structural requirement for the amino acid substrate. In general, tyrosine derivatives
with small substituents ortho- to the
d
OH group of tyrosine are tolerated by the
enzyme (
Coudijzer & Joniau, 1990; Joniau, Coudijzer, & Decuyper, 1990; Kalisz,
Erck, Plessmann & Wehland, 2000; Monasterio, Nova, Lopezbrauet & Lagos,
1995
). At least two of these tyrosine derivatives possess reactive functional groups
suitable for bioorthogonal chemical labeling. The tyrosine analog 3-azidotyrosine
has been used as a photoinhibitor of TTL. Conceivably, tubulin containing
3-azidotyrosine can be labeled using “click” chemistry with alkyne-containing
reagents, many of which are commercially available. This possibility has not been
demonstrated in the literature, and we have not attempted it in our lab. Instead,
we have synthesized and studied the tyrosine derivative 3fY. We selected a formyl
group rather than the more commonly used acyl group for the 3-position because of
its smaller size and also because of the higher reactivity of aldehydes with nucleo-
philes versus ketones with nucleophiles. The tyrosine derivative is a poorer substrate
than tyrosine for the ligation reaction, but complete retyrosination can be achieved
with 3fY, given sufficient time and substrate concentration.
The final component of the first part of the procedure is TTL. We use a recom-
binant human TTL with a GST tag, prepared from a commercial vector. We have
found that it is not necessary to remove the GST from the enzyme; the procedures
described here are for the TTL-GST fusion protein. This enzyme can be stored
at
D
50
C for many months without observable loss of activity.
The second part of the protocol is the bioorthogonal ligation reaction. In the pro-
cedures described here, the probe of interest possesses a hydrazide or an aromatic
hydrazine as the reactive functional group. Few aromatic hydrazine-containing
probes are commercially available at this time, but many different hydrazide probes
can be purchased. In recent years, it has become common to use a hydroxylamine-
containing probe as the nucleophile in these coupling reactions (
Dirksen, Hackeng, &
Dawson, 2006
). Owing to the similar reactivity of the nucleophiles, it is likely that a
hydroxylamine-containing probe can be successfully substituted for a hydrazide probe
in these procedures.
Coupling reactions between hydrazides, hydrazines, or hydroxylamines and
aldehydes are typically fastest at acidic pH: pH 4.5 and lower (
Cordes & Jencks,
1962
). The rate of the reaction drops sharply with increasing pH. Optimally, the cou-
pling reaction should be performed under conditions that retain the activity of the
protein, and purified tubulin is most stable near neutral pH and at cold temperatures.
