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Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

View Article: PubMed Central - PubMed

ABSTRACT

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology.

No MeSH data available.


Fluorophore-conjugated multinuclear Zn2+ complexes (Zn(II)-DpaTyrs): (a) FITC-binuclear Zn(II)-DpaTyr, (b) FITC-tetranuclear Zn(II)-DpaTyr, (c) Cy5-tetranuclear Zn(II)-DpaTyr, (d) SNARF-binuclear Zn(II)-DpaTyr, (e) pyrene-binuclear Zn(II)-DpaTyr, (f) N-α-chloroacetyl-rhodamine-binuclear Zn(II)-DpaTyr
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f6-sensors-08-01004: Fluorophore-conjugated multinuclear Zn2+ complexes (Zn(II)-DpaTyrs): (a) FITC-binuclear Zn(II)-DpaTyr, (b) FITC-tetranuclear Zn(II)-DpaTyr, (c) Cy5-tetranuclear Zn(II)-DpaTyr, (d) SNARF-binuclear Zn(II)-DpaTyr, (e) pyrene-binuclear Zn(II)-DpaTyr, (f) N-α-chloroacetyl-rhodamine-binuclear Zn(II)-DpaTyr

Mentions: Another peptide tag/artificial probe system, different from tetracysteine/biarsencal or His-tag/NTA systems, was also reported. Hamachi et al. developed a protein labeling system composed of a oligoaspartate tag (D4 tag, (D4)n, n = 1-3) and multinuclear Zn2+ complexes (Zn(II)-DpaTyrs); FITC-conjugated binuclear complex (Figure 6(a)) and FITC or Cy5-conjugated tetranuclear complexes (Figure 6(b),(c)) [54]. Higher affinity was observed with increasing the number of Asp in D4 tag (binding constants: D2; 7.1×103 M-1, D3; 6.3×104 M-1, D4; 6.9×105 M-1, D5; 8.6×105 M-1 (comparable to D4) for FITC-conjugated binuclear complex). In addition, the moderate binding affinity between FITC-conjugated binuclear complex and D4-RNase (1.2 × 104 M-1) was significantly enhanced when FITC-conjugated tetranuclear complex and RNase fused with a longer tag (D4-G-D4) were used alternatively (1.8 × 107 M-1). Fluorescent labeling of muscarinic acetylcholine receptor (mlAChR) by the tetranuclear complexes showed that the complexes did not interfere with the original activity of the receptor or the evoked intracellular signaling.


Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology
Fluorophore-conjugated multinuclear Zn2+ complexes (Zn(II)-DpaTyrs): (a) FITC-binuclear Zn(II)-DpaTyr, (b) FITC-tetranuclear Zn(II)-DpaTyr, (c) Cy5-tetranuclear Zn(II)-DpaTyr, (d) SNARF-binuclear Zn(II)-DpaTyr, (e) pyrene-binuclear Zn(II)-DpaTyr, (f) N-α-chloroacetyl-rhodamine-binuclear Zn(II)-DpaTyr
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3927527&req=5

f6-sensors-08-01004: Fluorophore-conjugated multinuclear Zn2+ complexes (Zn(II)-DpaTyrs): (a) FITC-binuclear Zn(II)-DpaTyr, (b) FITC-tetranuclear Zn(II)-DpaTyr, (c) Cy5-tetranuclear Zn(II)-DpaTyr, (d) SNARF-binuclear Zn(II)-DpaTyr, (e) pyrene-binuclear Zn(II)-DpaTyr, (f) N-α-chloroacetyl-rhodamine-binuclear Zn(II)-DpaTyr
Mentions: Another peptide tag/artificial probe system, different from tetracysteine/biarsencal or His-tag/NTA systems, was also reported. Hamachi et al. developed a protein labeling system composed of a oligoaspartate tag (D4 tag, (D4)n, n = 1-3) and multinuclear Zn2+ complexes (Zn(II)-DpaTyrs); FITC-conjugated binuclear complex (Figure 6(a)) and FITC or Cy5-conjugated tetranuclear complexes (Figure 6(b),(c)) [54]. Higher affinity was observed with increasing the number of Asp in D4 tag (binding constants: D2; 7.1×103 M-1, D3; 6.3×104 M-1, D4; 6.9×105 M-1, D5; 8.6×105 M-1 (comparable to D4) for FITC-conjugated binuclear complex). In addition, the moderate binding affinity between FITC-conjugated binuclear complex and D4-RNase (1.2 × 104 M-1) was significantly enhanced when FITC-conjugated tetranuclear complex and RNase fused with a longer tag (D4-G-D4) were used alternatively (1.8 × 107 M-1). Fluorescent labeling of muscarinic acetylcholine receptor (mlAChR) by the tetranuclear complexes showed that the complexes did not interfere with the original activity of the receptor or the evoked intracellular signaling.

View Article: PubMed Central - PubMed

ABSTRACT

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology.

No MeSH data available.