GABAC Receptor Binding of Quantum-Dot Conjugates of Variable Ligand Valency
| Autor: | David R. Pepperberg, Haohua Qian, Deborah M. Little, Hélène A. Gussin, Sandra J. Rosenthal, Ian D. Tomlinson, Niraj J. Muni |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Stereochemistry
Biomedical Engineering Pharmaceutical Science Bioengineering macromolecular substances Sulfides Conjugated system Ligands Article Fluorescence Polyethylene Glycols GABAA-rho receptor Methylamines chemistry.chemical_compound Receptors GABA Quantum Dots PEG ratio Cadmium Compounds Binding site Selenium Compounds Pharmacology Binding Sites Molecular Structure Muscimol Chemistry Organic Chemistry technology industry and agriculture Valency Ligand (biochemistry) Zinc Compounds Biotechnology Conjugate |
| Zdroj: | Bioconjugate Chemistry. 21:1455-1464 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc100050s |
| Popis: | Highly fluorescent CdSe quantum dots (qdots) can serve as a platform for tethering multiple copies of a receptor-targeted ligand, affording study of how the level of multivalency affects receptor binding. We previously showed that qdots conjugated with long PEG chains terminated by muscimol, a known GABA(C) agonist, exhibit specific binding to the surface membrane of GABA(C) receptor-expressing Xenopus oocytes. The present report addresses the effect of varying the number, i.e., valency, of muscimol- (M-) terminated PEG chains attached to the qdot on binding of the resulting conjugate to GABA(C) receptors. M-PEG-qdots of differing muscimol valency were prepared by conjugating AMP-CdSe/ZnS qdots with muscimol-terminated and methylamine-terminated PEG chains in proportions designed to yield varying percentages of muscimol-terminated chains among the total approximately 150-200 chains bound to the qdot. The investigated valencies represented 0%, approximately 25%, approximately 50%, and 100% loading with muscimol (preparations termed M-PEG-qdot0, M-PEG-qdot25, M-PEG-qdot50, and M-PEG-qdot100, respectively. Binding of a given conjugate to surface membranes of GABA(C) receptor-expressing oocytes was analyzed by quantitative fluorescence microscopy following defined incubation with approximately 30 nM of the conjugate. With 5-20 min incubation, the fluorescence signal resulting from incubation with M-PEG-qdot25 exceeded, by approximately 6-fold, the fluorescence level obtained with M-PEG-qdot preparations that lacked muscimol-terminated chains (M-PEG-qdot0). M-PEG-qdot50 yielded a net signal roughly similar to that of M-PEG-qdot25, and that produced by M-PEG-qdot100 exceeded, by approximately 30-50%, those for M-PEG-qdot25 and M-PEG-qdot50. The time course of changes in oocyte surface membrane fluorescence resulting from the introduction of and removal of M-PEG-qdots in the medium bathing the oocyte indicated only a modest dependence of both binding and wash-out kinetics on muscimol valency. The results demonstrate a dependence of the binding activity of the M-PEG-qdot conjugates on muscimol valency, presumably reflecting higher GABA(C) avidity and/or affinity of the muscimol at high valency, and provide insight on the interactions of membrane receptor proteins with qdot conjugates containing multiple copies of a receptor-targeting ligand. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|