C O M M U N I C A T I O N S
Figure 5. Covalent labeling of CA6D4-MBP or C-MBP in E. coli lysate
(a) and inside E. coli cell (c) with 3-2Zn(II). Reaction conditions: 20 µM
3-2Zn(II) in HBS buffer, pH 7.4, 4 °C. (a, c) SDS-PAGE analysis of the
labeling reaction in E. coli lysate overexpressing of CA6D4-MBP (lane 1
and 3) or C-MBP (lane 2 and 4). (b) confocal laser scanning (left) and
differential interference contrast (DIC, right) images of E. coli expressing
CA6D4-MBP incubated with 3-2Zn(II).
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Figure 4. (a, b) SDS-PAGE analysis of the covalent labeling of CA6D4-
EGFP with 2-2Zn(II) in the protein mixture. The Coomassie staining (left)
and in-gel fluorescence visualization (right) were performed after Huisgen
reaction with coumarin azide 4: BSA, bovine serum albumin; GST,
glutathione S-transferase; HG, hemoglobin; TR, thioredoxin; â-Gal, â-ga-
lactosidase; OVA, ovalbumin; RNase, ribonuclease. Reaction conditions:
20 µM 2-2Zn(II), 5 µM CA6D4-EGFP in 50 mM HEPES, 100 mM NaCl,
1 mM DTT, pH 7.2, 20 °C.
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site-specific covalent labeling of proteins. The present method using
a genetically encodable reactive tag and the small molecular probe
is potentially applicable to a wide variety of proteins, thus would
provide a general tool for protein researches such as bioimaging,
protein engineering, and biophysical studies of protein structure
and dynamics.
102, 4639.
(7) The observed increase of the mass number 755 is smaller than the
theoretical value 986 (2-2Zn(II)-HCl-3Cl). This shortage probably comes
from the decomplexation of the Zn(II) ion(s) and/or the cleavage of the
dipicolylamine (Dpa) fragment from the probe owing to the acidic
pretreatment of the sample for desalting and harsh laser-induced ionization
of the labeled protein in the MS analysis. The same shortage was observed
in MALDI-TOF mass analysis of 1-2Zn(II).
(8) Bra¨se, S.; Gil, C.; Knepper, K.; Zimmermann, V. Angew. Chem., Int. Ed.
2005, 44, 5188.
Acknowledgment. This study was supported by research grants
from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan, and the Japan Society for the Promotion of
Science.
(9) The observed fluorescence only derives from the coumarin derivative 4
introduced into EGFP and not from EGFP. During the pretreatment for
SDS-PAGE (95 °C, 3min), EGFP completely lost its fluorescence.
(10) The labeling reaction of 3-2Zn(II) with the non overexpressed EGFP
having the CA6D4 tag (<1% of total proteins) did not proceed in E. coli
lysate, which is probably due to the competitive inhibition by phosphate
derivatives such as ATP and the moderate binding affinity of the present
tag-probe pair (Kd ) 1.4 µM).
Supporting Information Available: Synthesis and characterization
of the compounds, and experimental details. This material is available
JA074176D
9
J. AM. CHEM. SOC. VOL. 129, NO. 51, 2007 15779