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Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins.

ABSTRACT: Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane-bound proteins which are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcoreTM, have been adapted for membrane protein analysis by building supported lipid layers or capturing lipid vesicles on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of G protein coupled receptor ligands and applications in basic cellular biology.

SUBMITTER: Maynard JA

PROVIDER: S-EPMC2790208 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins.

Maynard Jennifer A JA Lindquist Nathan C NC Sutherland Jamie N JN Lesuffleur Antoine A Warrington Arthur E AE Rodriguez Moses M Oh Sang-Hyun SH

Biotechnology journal 20091101 11

Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane ...[more]

PMID: 19918786

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Project description:BackgroundSurface plasmon resonance imaging (SPRI) is a label-free technique that can image refractive index changes at an interface. We have previously used SPRI to study the dynamics of cell-substratum interactions. However, characterization of spatial resolution in 3 dimensions is necessary to quantitatively interpret SPR images. Spatial resolution is complicated by the asymmetric propagation length of surface plasmons in the x and y dimensions leading to image degradation in one direction. Inferring the distance of intracellular organelles and other subcellular features from the interface by SPRI is complicated by uncertainties regarding the detection of the evanescent wave decay into cells. This study provides an experimental basis for characterizing the resolution of an SPR imaging system in the lateral and distal dimensions and demonstrates a novel approach for resolving sub-micrometer cellular structures by SPRI. The SPRI resolution here is distinct in its ability to visualize subcellular structures that are in proximity to a surface, which is comparable with that of total internal reflection fluorescence (TIRF) microscopy but has the advantage of no fluorescent labels.ResultsAn SPR imaging system was designed that uses a high numerical aperture objective lens to image cells and a digital light projector to pattern the angle of the incident excitation on the sample. Cellular components such as focal adhesions, nucleus, and cellular secretions are visualized. The point spread function of polymeric nanoparticle beads indicates near-diffraction limited spatial resolution. To characterize the z-axis response, we used micrometer scale polymeric beads with a refractive index similar to cells as reference materials to determine the detection limit of the SPR field as a function of distance from the substrate. Multi-wavelength measurements of these microspheres show that it is possible to tailor the effective depth of penetration of the evanescent wave into the cellular environment.ConclusionWe describe how the use of patterned incident light provides SPRI at high spatial resolution, and we characterize a finite limit of detection for penetration depth. We demonstrate the application of a novel technique that allows unprecedented subcellular detail for SPRI, and enables a quantitative interpretation of SPRI for subcellular imaging.

Dataset Information

Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins.

Publications

Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins.

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