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Microplate-based characterization of protein-phosphoinositide binding interactions using a synthetic biotinylated headgroup analogue.

ABSTRACT: Membrane lipids act as important regulators of a litany of important physiological and pathophysiological events. Many of them act as site-specific ligands for cytosolic proteins in binding events that recruit receptors to the cell surface and control both protein function and subcellular localization. Phosphatidylinositol phosphates (PIP(n)s) are a family of signaling lipids that regulate numerous cellular processes by interacting with a myriad of protein binding modules. Characterization of PIP(n)-binding proteins has been hampered by the lack of a rapid and convenient quantitative assay. Herein, microplate-based detection is presented as an effective approach to characterizing protein-PIP(n) binding interactions at the molecular level. With this assay, the binding of proteins to isolated PIP(n) headgroups is detected with high sensitivity using a platform that is amenable to high-throughput screening. In the studies described herein, biotinylated PI-(4,5)-P(2) headgroup analogue 1 was designed, synthesized, and immobilized onto 96-well streptavidin-coated microplates to study receptor binding. This assay was used to characterize the binding of the PH domain of beta-spectrin to this headgroup. The high affinity interaction that was detected for surface association (K(d, surf) = 6 nM +/- 3), demonstrates that receptor binding modules can form high affinity interactions with lipid headgroups outside of a membrane environment. The results also indicate the feasibility of the assay for rapid characterization of PIP(n)-binding proteins as well as the promise for high-throughput analysis of protein-PIP(n) binding interactions. Finally, this assay was also employed to characterize the inhibition of the binding of receptors to the PIP(n)-derivatized microplates using solution phase competitors. This showcases the viability of this assay for rapid screening of inhibitors of PIP(n)-binding proteins.

SUBMITTER: Gong D

PROVIDER: S-EPMC2656446 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Microplate-based characterization of protein-phosphoinositide binding interactions using a synthetic biotinylated headgroup analogue.

Gong Denghuang D Smith Matthew D MD Manna Debasis D Bostic Heidi E HE Cho Wonhwa W Best Michael D MD

Bioconjugate chemistry 20090201 2

Membrane lipids act as important regulators of a litany of important physiological and pathophysiological events. Many of them act as site-specific ligands for cytosolic proteins in binding events that recruit receptors to the cell surface and control both protein function and subcellular localization. Phosphatidylinositol phosphates (PIP(n)s) are a family of signaling lipids that regulate numerous cellular processes by interacting with a myriad of protein binding modules. Characterization of PI ...[more]

PMID: 19182890

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Project description:Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

Dataset Information

Microplate-based characterization of protein-phosphoinositide binding interactions using a synthetic biotinylated headgroup analogue.

Publications

Microplate-based characterization of protein-phosphoinositide binding interactions using a synthetic biotinylated headgroup analogue.

Similar Datasets

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