Synthesis of the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) and its use with the IAsys biosensor to study receptor-ligand interactions on model membranes.
This work describes the synthesis and use of the chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). This lipid is readily dispersed in aqueous media, both alone and when mixed with carrier lipids like dimyristoylphosphatidylcholine (DMPC). Fluorescence microscopic examination of membranes deposited from NTA-DTDA-containing liposomes shows that NTA-DTDA mixes uniformly with the carrier lipid, and does not phase separate. NTA-DTDA-membranes deposited onto the sensing surface of IAsys biosensor cuvettes show good stability, permitting use of the biosensor to study protein interactions. Hexahistidine-tagged proteins including recombinant forms of the extracellular regions of murine B7.1 (B7.1-6H) and of the human erythropoietin receptor (EPOR-6H) bind to NTA-DTDA-membranes; the stability of binding is dependent on both protein concentration, and density of NTA-DTDA. Kinetic measurements show that high stability of anchored proteins (t(1/2) approximately 10-20 h, apparent K(d) approximately 1 nM) can be achieved using membranes containing 25 mol% NTA-DTDA, but low levels of bound protein (<200 arc seconds). The system is used to study the interaction of human EPO with the EPOR anchored onto NTA-DTDA-membranes. In addition to the biological applications reported recently, the results show that NTA-DTDA can be a useful reagent in the study of receptor-ligand interactions.
Altin
J G
JG
School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, ACT 0200, Australia. joseph.altin@anu.edu.au
White
F A
FA
Easton
C J
CJ
D000588:Amines / Q000138:chemical synthesis*; D015374:Biosensing Techniques; D002614:Chelating Agents / Q000138:chemical synthesis*; D004134:Dimyristoylphosphatidylcholine; D004921:Erythropoietin / Q000737:chemistry; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000737:chemistry
D000588:Amines; D002614:Chelating Agents; D008024:Ligands; D008565:Membrane Proteins; D008567:Membranes, Artificial; D017467:Receptors, Erythropoietin; C405382:nitrilotriacetic acid ditetradecylamine; D004921:Erythropoietin; D009571:Nitrilotriacetic Acid; D004134:Dimyristoylphosphatidylcholine