Design of a multinuclear Zn(II) complex as a new molecular probe for fluorescence imaging of His-tag fused proteins.

A Zn(II) complex (Zn(II)-Ida) was designed as the new fluorescent probe for His-tag fused proteins. Thanks to the tight binding ability to histidine-rich sequences and bright fluorescence property of the Cy5-appended Zn(II)-Ida probes, selective and clear fluorescent imaging of the His-tag fused G-protein coupled receptors on live cell surfaces was carried out.

Selective covalent labeling of tag-fused GPCR proteins on live cell surface with a synthetic probe for their functional analysis.

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this paper, we report a covalent labeling method of tag-fused G-protein coupled receptor (GPCR) proteins expressing on cell surfaces utilizing small functional molecules. This method employs the selective and rapid reaction of a peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive alpha-chloroacetyl moiety. The covalent labeling of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) selectively proceeded under physiological conditions during short incubation (10-30 min) with Zn(II)-DpaTyr probes bearing various functional groups. Labeling with fluorophore-appended Zn(II)-DpaTyr probes enabled visualization of the GPCRs on the surface of HEK293 cells by fluorescence. Labeling with the biotin-appended probe allowed introduction of a biotin unit into the GPCRs. This biotin label was utilized for fluorescence bioimaging studies and postlabeling blotting analysis of the labeled GPCRs by use of the specific biotin-streptavidin interaction. The utility of this labeling method was demonstrated in several function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.

Binuclear Ni(II)-DpaTyr complex as a high affinity probe for an oligo-aspartate Tag tethered to proteins.

A complementary recognition pair of a short-peptide tag and a small molecular probe is a versatile molecular tool for protein detection, handling, and purification, and so forth. In this manuscript, we report that the binuclear Ni(II)-DpaTyr (DpaTyr=bis((dipicolylamino)methyl)tyrosine) complex serves as a strong binding probe for an oligo-aspartate tag tethered to a protein. Among various binuclear metal complexes of M-DpaTyr (M=Zn(II), Ni(II), Mn(II), Cu(II), Cd(II), Co(III), and Fe(III)), we have found that Ni(II)-DpaTyr (1-2Ni(II)) displays a strong-binding affinity (apparent binding constant: K(app) approximately 10(5) M(-1)) for an oligo-aspartate peptide under neutral aqueous conditions (50 mM HEPES, 100 mM NaCl, pH 7.2). Detailed isothermal-titration calorimetry (ITC) studies reveal that the tri-aspartate D3-tag (DDD) is an optimal sequence recognized by 1-2Ni(II) in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for Ni(II)- and Zn(II)-DpaTyr, show a negligible binding affinity for the oligo-aspartate peptide. The binding affinity was greatly enhanced in the pair between the dimer of Ni(II)-DpaTyr and the repeated D3 tag peptide (D3x2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K(app)=2x10(9) M(-1)) was achieved between the Ni(II)-DpaTyr dimer 4-4Ni(II) and the D3x2 tag peptide (DDDNGDDD). This affinity is approximately 100-fold stronger than that observed in the binding pair of the Zn(II)-DpaTyr (4-4Zn(II)) and the D4x2 tag (DDDDGDDDD), a useful tag-probe pair previously reported by us. The recognition pair of the Ni(II)-DpaTyr probe and the D3x2 tag can also work effectively on a protein surface, that is, 4-4Ni(II) is strongly bound to the FKBP12 protein tethered with the D3x2 tag (DDDNGDDD) with a large K(app) value of 5x10(8) M(-1). Taking advantage of the strong-binding affinity, this pair was successfully applied to the selective inactivation of the tag-fused beta-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.

FLAG-tag selective covalent protein labeling via a binding-induced acyl-transfer reaction.

A FLAG tag selective protein labeling method is newly developed in this study. Coupling of the selective binding between synthetic Ni-complex probe and FLAG tag with the acyl transfer reaction enables the site-selective covalent modification of FLAG peptide and FLAG-tag fused protein.

Site-specific covalent labeling of His-tag fused proteins with a reactive Ni(II)-NTA probe.

A new method for covalent labeling of a His-tag fused protein with a small reactive probe was developed; this method is based on the complementary interaction between the His-tag and Ni(II)-NTA, which facilitates a nucleophilic reaction between a histidine residue of the tag and the electrophilic tosyl group of the Ni(II)-NTA probe by the proximity effect.