Communications
[
6]
tured by both photo-crosslinkers. Both molecules captured a
distinct product of approximately 80 kDa, consistent with the
sodium ascorbate at 378C (Figure 3A). After 2 hours, the
biotin-conjugated proteins were isolated on neutravidin mag-
netic beads and analyzed by western blot probing for the
Myc-tagged Gal80 species (Figure 3B). With this strategy, the
Lexa+Gal4–Gal80 complex is only observed for BPKyne-incor-
porated proteins that have been irradiated and functionalized
with the biotin-azide probe, thus demonstrating the ability of
the bioorthogonal alkyne handle to be specifically labeled. As
a comparison, traditional immunological techniques were used
to isolate the pBpa-containing Gal80–Gal4 crosslinked com-
plex, which was visualized by western blot. Importantly, when
visualized by western blot with the Myc-HRP antibody, less
background is seen when BPKyne-containing samples isolated
through CuAAC and neutravidin pull-down are compared to
pBpa-containing proteins immunoprecipitated with Myc; this
results in nonspecific isolation of all protein containing an en-
dogenous Myc epitope (Figures 3B and S2). These experiments
illustrate the advantages of the bifunctional BPKyne molecule,
which captures specific PPIs upon irradiation and allows them
to be isolated from their cellular environment post-functionali-
zation.
[
4a]
Gal4–Gal80 complex (Figure 2C).
To confirm this, a Myc6-
tagged Gal80 construct was transformed into live yeast with
the UAA-incorporated LexA+Gal4 fusion protein. After irradia-
tion, lysis, and western blot analysis probing for the Myc-
tagged Gal80 protein, both pBpa and BPKyne captured a Gal4–
Gal80 crosslinked product (Figure 2D). When comparing the
amount of crosslinked Gal4–Gal80, BPKyne’s crosslinking effi-
ciency is approximately two-thirds that of the parent molecule,
at least in this context. A small decrease was expected due to
[
11]
the stereoelectronic influence of the triple bond (Figure 2C
and D).
Once incorporation and crosslinking were confirmed, the
bioconjugation capability of BPKyne was characterized post-
crosslinking and was compared to that of traditional immuno-
[4a]
logical methods for the isolation of crosslinked products.
The UAA was incorporated into a Gal80 construct with posi-
tion 245 mutated to the amber stop codon. This site is located
at the outer edge of the Gal4 binding interface, therefore
when this construct is irradiated only the Gal80–Gal4 complex
should be captured; this allows a single interaction to be vi-
sualized. To demonstrate that the bioorthogonal alkyne handle
of BPKyne could be functionalized post-crosslinking, live yeast
expressing Flag-tagged LexA+Gal4 and Myc-tagged Gal80
with pBpa or BPKyne incorporated at position 245 was grown
under glucose conditions and irradiated to capture the Gal80–
Here we have demonstrated the first incorporation of the bi-
functional UAA BPKyne into live yeast cells by using the E. coli
tyrosyl tRNA/synthetase system and have illustrated the utility
of BPKyne for the isolation of crosslinked products from their
native environment. Utilizing the Gal4 and Gal80 yeast proteins
we have shown that BPKyne is incorporated with similar ex-
pression yields to pBpa without requiring further mutagenesis.
Along with the similar crosslinking yield compared to pBpa, we
have illustrated that BPKyne-containing proteins can be isolat-
ed from whole-cell lysate after functionalization with a biotiny-
lated azide probe. Although we used western blotting for visu-
alization in this proof-of-principle study, mass spectrometry
could also be used to characterize isolated crosslinked adducts.
This strategy also enables the capture and isolation of PPIs for
which antibodies are not efficient or available or when geneti-
cally encoded epitope tags, such as Myc or Flag, cannot be
appended without impairing protein structure or function. The
bioorthogonal alkyne handle enables the direct labeling of
crosslinked PPIs of interest, which will be particularly advanta-
geous in the discovery of novel PPIs.
Gal4 binding event. After lysis, biotin-PEG -azide was conjugat-
3
ed to the BPKyne-incorporated Gal80 species through a Huis-
gen cycloaddition in whole-cell lysate by using copper(II) sul-
fate, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and
Acknowledgements
We are grateful to the NSF CHE 1412759 for support of this
work. We thank Professor Brent Martin and Dr. Thomas Custer
for insightful discussion and Dr. Amanda Dugan for construction
of the LexA+Gal4 WT-5X Flag LexA+Gal4 849TAG-5X Flag ex-
pression plasmids.
Figure 3. Analysis of BPKyne bioconjugation by CuAAC. A) Experimental
workflow for isolation of Gal80 245BPKyne–Gal4 crosslinked products from
yeast cells. B) Biotinylation of Gal4–Gal80 crosslinked product through
CuAAC cycloaddition. The BPKyne-incorporated Gal80–Gal4 crosslinked
product was isolated from solution by using CuAAC and neutravidin mag-
netic beads and analyzed by western blot (a-Myc). The Gal4–Gal80 cross-
linked product is only isolated in the presence of BPKyne and UV when con-
jugated to the biotin probe. (See Figure S4 for expression of Myc-Gal80
Keywords: bioorthogonal labeling · click chemistry · photo-
crosslinking · protein–protein interactions · unnatural amino
acids
245UAA.)
ChemBioChem 2016, 17, 1 – 5
3
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ
Joiner, Cassandra M.
