PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Djoshkun Shengjuler; Simou Sun; Paul S. Cremer; Craig E. Cameron

doi:10.3791/55869

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Method Article

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

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DOI:

10.3791/55869

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July 27th, 2017

Djoshkun Shengjuler¹ , Simou Sun² , Paul S. Cremer¹^,² , Craig E. Cameron¹

¹Department of Biochemistry and Molecular Biology, The Pennsylvania State University, ²Department of Chemistry, The Pennsylvania State University

Summary

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Abstract

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-δ1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) interaction as an example. The apparent dissociation constant (K_d,app) for this interaction was 0.39 ± 0.05 µM, similar to K_d,app values obtained by others. As previously observed, the PLC-δ1 PH domain is PI(4,5)P₂ specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein's capacity to interact with membranes.

Introduction

Myriad interactions and biochemical processes take place on two-dimensionally fluid membrane surfaces. Membrane-enclosed organelles in eukaryotic cells are unique not only in biochemical processes and their associated proteome but also in their lipid composition. One exceptional class of phospholipids is phosphoinositides (PIPs). Even though they comprise only 1% of the cellular lipidome, they play a crucial role in signal transduction, autophagy, and membrane trafficking, among others¹^,²^,³^,⁴. Dynamic phosphorylation of the inositol head gro....