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Detecting protein-ligand binding on supported bilayers by local pH modulation.

ABSTRACT: Herein, we describe a highly sensitive technique for detecting protein-ligand binding at the liquid/solid interface. The method is based upon modulation of the interfacial pH when the protein binds. This change is detected by ortho-Texas Red DHPE, which is doped into supported phospholipid bilayers and used as a pH-sensitive dye. The dye molecule fluoresces strongly at acidic pH values but not basic ones and has an apparent pK(A) of 7.8 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes containing 0.5 mol % biotin-cap-PE. This method was used to detect antibiotin/biotin binding interactions as well as the binding of cholera toxin B subunits to GM(1). Since these proteins are negatively charged under the conditions of the experiment the interface became slightly more acidic upon binding. In each case, the equilibrium dissociation constant was determined by following the rise in fluorescence as protein was introduced. This change is essentially linear with protein coverage under the conditions employed. For the biotin/antibiotin system it was determined that K(D) = 24 +/- 5 nM, which is in excellent agreement with classical measurements made by total internal reflection fluorescence microscopy involving fluorophore-conjugated antibody molecules. Moreover, the limit of detection was approximately 350 fM at the 99% confidence level. This corresponds to 1 part in 69,000 of the K(D) value. Such a finding compares favorably with surface plasmon resonance studies of similar systems and conditions. The assay could be run in imaging mode to obtain multiple simultaneous measurements using a CCD camera.

SUBMITTER: Jung H

PROVIDER: S-EPMC3195364 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Detecting protein-ligand binding on supported bilayers by local pH modulation.

Jung Hyunsook H Robison Aaron D AD Cremer Paul S PS

Journal of the American Chemical Society 20090101 3

Herein, we describe a highly sensitive technique for detecting protein-ligand binding at the liquid/solid interface. The method is based upon modulation of the interfacial pH when the protein binds. This change is detected by ortho-Texas Red DHPE, which is doped into supported phospholipid bilayers and used as a pH-sensitive dye. The dye molecule fluoresces strongly at acidic pH values but not basic ones and has an apparent pK(A) of 7.8 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membran ...[more]

PMID: 19125648

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Project description:The development of model systems that mimic biological interactions and allow the control of both receptor and ligand densities, is essential for a better understanding of biomolecular processes, such as the recruitment of receptors at interfaces, at the molecular level. Here we report a model system based on supported lipid bilayers (SLBs) for the investigation of the clustering of receptors at their interface. Biotinylated SLBs, used as cell membrane mimics, were functionalized with streptavidin (SAv), used here as receptor. Subsequently, biotinylated small (SUVs) and giant (GUVs) unilamellar vesicles were bound to the SAv-functionalized SLBs by multivalent interactions and found to induce the recruitment of both SAv on the SLB surface and the biotin moieties in the vesicles. The recruitment of receptors was investigated with quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed the identification of the biotin and SAv densities necessary to obtain receptor recruitment. At approx. 0.6% of biotin in the vesicles, a transition between dense and low vesicle packing was observed, which coincided with the transitions between recruitment in the vesicles vs. recruitment in the SLB and between full and partial use of the biotin moieties in the vesicle. Direct optical visualization of the clustering at the interface of individual GUVs with the SLB platform was achieved with fluorescence microscopy, showing recruitment of SAv at the contact area as well as the deformation of the vesicles upon binding. Different vesicle binding regimes were observed for lower and higher biotin densities in the vesicles and at the SLBs. A more quantitative analysis of the molecular parameters implied in the interaction, indicated that approx. 10% of the vesicle area constitutes the contact area. Moreover, the SUV binding and recruitment appeared to be fast on the analysis time scale, whereas the binding of GUVs is slower due to the larger SLB area over which SAv recruitment needs to occur. The mechanisms revealed in this study may provide insight in biological processes in which recruitment occurs.

Dataset Information

Detecting protein-ligand binding on supported bilayers by local pH modulation.

Publications

Detecting protein-ligand binding on supported bilayers by local pH modulation.

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