carbocyanines and nickel-nitrilotriacetic-acid

The hexahistidine (His(6))/nickel(II)-nitrilotriacetic acid (Ni(2+)-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His(6) proteins. Here we explore several applications of the NTA derivative, (Ni(2+)-NTA)(2)-Cy3. This molecule binds our two model His(6) proteins, N-ethylmaleimide sensitive factor (NSF) and O(6)-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K approximately 1.5 x 10(6) M(-1)) and no effect on their activity. Its high specificity makes (Ni(2+)-NTA)(2)-Cy3 ideal for detecting His(6) proteins in complex mixtures of other proteins, allowing (Ni(2+)-NTA)(2)-Cy3 to be used as a probe in crude cell extracts and as a His(6)-specific gel stain. (Ni(2+)-NTA)(2)-Cy3 binding is reversible in 10mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (k(exchange) approximately 5 x 10(-6) s(-1) with 0.2 microM labeled protein in the presence of 1 microM His(6) peptide). Labeling with (Ni(2+)-NTA)(2)-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni(2+)-NTA)(2)-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.

Topics: Alkyl and Aryl Transferases; Carbocyanines; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Histidine; N-Ethylmaleimide-Sensitive Proteins; Nitrilotriacetic Acid; Oligopeptides; Organometallic Compounds; Protein Interaction Domains and Motifs; Spectrometry, Fluorescence; Ultracentrifugation

Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (*30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue "Stains-all" dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli.

Topics: 4-Chloro-7-nitrobenzofurazan; Calcium; Carbocyanines; Chromatography, Affinity; Dithionite; Escherichia coli; Gene Expression; Humans; Inclusion Bodies; Nitrilotriacetic Acid; Organometallic Compounds; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipid Transfer Proteins; Protein Folding; Proteolipids; Recombinant Proteins; Solubility; Spectrometry, Fluorescence; Urea