PMID- 19499918
OWN - NLM
STAT- MEDLINE
DCOM- 20090915
LR  - 20191210
IS  - 1520-5126 (Electronic)
IS  - 0002-7863 (Linking)
VI  - 131
IP  - 25
DP  - 2009 Jul 1
TI  - Quenched ligand-directed tosylate reagents for one-step construction of turn-on 
      fluorescent biosensors.
PG  - 9046-54
LID - 10.1021/ja902486c [doi]
AB  - Semisynthetic fluorescent biosensors consisting of a protein framework and a 
      synthetic fluorophore are powerful analytical tools for specific detection of 
      biologically relevant molecules. We report herein a novel method that allows for 
      the construction of turn-on fluorescent semisynthetic biosensors in a one-step 
      manner. The strategy is based on the ligand-directed tosyl (LDT) chemistry, a new 
      type of affinity-guided protein labeling scheme which can site-specifically 
      introduce synthetic probes to the surface of proteins with concomitant release of 
      the affinity ligands. Novel quenched ligand-directed tosylate (Q-LDT) reagents 
      were designed by connecting an organic dye to a conjugate of a protein ligand and 
      a fluorescence quencher through a tosyl linker. The Q-LDT-mediated labeling 
      directly converts a natural protein to a fluorescently labeled protein that 
      remains noncovalently complexed with the cleaved ligand-tethered quencher. The 
      fluorescence of this labeled protein is initially quenched and only in the 
      presence of specific analytes is the fluorescence enhanced (turned on) due to the 
      expulsion of the ligand-quencher fragment. Using a single labeling step, this 
      approach was successfully applied to carbonic anhydrase II (CAII) and a Src 
      homology 2 (SH2) domain to generate turn-on fluorescent biosensors toward CAII 
      inhibitors and phosphotyrosine peptides, respectively. Detailed investigations 
      revealed that the obtained biosensors exhibit their natural ligand selectivity. 
      The high target-specificity of the LDT chemistry also allowed us to prepare the 
      SH2 domain-based biosensor not only in a purified form but also in a bacterial 
      cell lysate. These results demonstrate the utility of the Q-LDT-based approach to 
      expand the applications of semisynthetic biosensors.
FAU - Tsukiji, Shinya
AU  - Tsukiji S
AD  - Department of Synthetic Chemistry and Biological Chemistry, Graduate School of 
      Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
FAU - Wang, Hangxiang
AU  - Wang H
FAU - Miyagawa, Masayoshi
AU  - Miyagawa M
FAU - Tamura, Tomonori
AU  - Tamura T
FAU - Takaoka, Yousuke
AU  - Takaoka Y
FAU - Hamachi, Itaru
AU  - Hamachi I
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Am Chem Soc
JT  - Journal of the American Chemical Society
JID - 7503056
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Ligands)
RN  - 0 (Phosphopeptides)
RN  - 0 (Tosyl Compounds)
RN  - 21820-51-9 (Phosphotyrosine)
RN  - EC 4.2.1.- (Carbonic Anhydrase II)
SB  - IM
MH  - Amino Acid Sequence
MH  - Biosensing Techniques/*methods
MH  - Carbonic Anhydrase II/*antagonists & inhibitors/metabolism
MH  - Enzyme Inhibitors/*analysis/metabolism
MH  - Escherichia coli/chemistry/genetics
MH  - Fluorescent Dyes/chemical synthesis/*chemistry/metabolism
MH  - Humans
MH  - Ligands
MH  - Models, Molecular
MH  - Phosphatidylinositol 3-Kinases/chemistry/genetics/metabolism
MH  - Phosphopeptides/*analysis/metabolism
MH  - Phosphotyrosine/analysis/metabolism
MH  - Protein Binding
MH  - Protein Structure, Tertiary
MH  - Tosyl Compounds/chemical synthesis/*chemistry/metabolism
EDAT- 2009/06/09 09:00
MHDA- 2009/09/16 06:00
CRDT- 2009/06/09 09:00
PHST- 2009/06/09 09:00 [entrez]
PHST- 2009/06/09 09:00 [pubmed]
PHST- 2009/09/16 06:00 [medline]
AID - 10.1021/ja902486c [doi]
PST - ppublish
SO  - J Am Chem Soc. 2009 Jul 1;131(25):9046-54. doi: 10.1021/ja902486c.