Abstract

The identification of epitopes involved in protein-protein interactions is essential for understanding protein structure and function. Large scale efforts, although identifying the interactions, did not always yield these epitopes, could not confirm most of the known interactions, and seemed particularly unsuccessful for native intrinsic membrane proteins. We have developed a fluidics-based approach (non-steady-state kinetics) to obtain the broadest set of the epitopes interacting with a given target and applied it to a phage display methodology optimized for membrane proteins. Phages expressing a liver cDNA library were screened against a membrane protein (voltage-dependent anion channel) reconstituted into liposomes and captured on a chip surface. The controlled fluidics was obtained by a surface plasmon resonance (SPR) device that combined the advantages of working with minute reaction volumes and non-equilibrium conditions. We demonstrated selective enrichment of binders and could even select for different binding affinities by fractionation of the selected outputs at various elution times. With voltage-dependent anion channel as bait (a mitochondrial channel critical for cellular metabolism and apoptosis) we found at least 40% of its already reported ligands and independently confirmed 55 novel functional interactions, some of which fully blocked the channel. This highly efficient approach is generally applicable for any protein and could be automated and scaled up even without the use of a SPR device. The epitopes directly identified by this method are useful not only for unraveling interactomes but also for drug design and therapeutics.

Highlights

  • The identification of epitopes involved in protein-protein interactions is essential for understanding protein structure and function

  • Our strategy combined (i) exploring the full set of the protein epitopes expressed in a human liver cell line, (ii) using the native fold of the membrane protein achieved by its successful functional incorporation into liposomes, and (iii) the high stability, small surface, and controlled fluidics obtained by using a surface plasmon resonance (SPR)1 detector device

  • The purified target protein was embedded into large unilamellar vesicles (LUVs), and to obtain the “native” conformation of the protein, we used conditions known for many years to allow the functional reconstitution of the voltage-dependent anion channel (VDAC) protein (28 –30)

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Summary

EXPERIMENTAL PROCEDURES

Phage Selection—To select for phages that bind to VDAC, the initial library was amplified and injected at very low flow rate (1 ␮l/min) over a chip covered with VDAC liposomes (ϳ5000-RU level). Such a low flow rate allows a better competition between the viral particles for the binding sites. To that effect the sensor chip was first washed thoroughly by injection of buffer at high flow rate (90 ␮l at 30 ␮l/min) followed by an injection of 1% SDS (6 ␮l at 2 ␮l/min) to remove phages and liposomes from the sensor surface. Slopes during the reswelling phase of the experiments were computed by linear fits

RESULTS
Method used
DISCUSSION

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