Hunting Interactomes of a Membrane Protein

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 (voltagedependent 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. Molecular & Cellular Proteomics 5:1667-1680, 2006.The sequencing of genomes led to the study of interactomes, i.e. the context-dependent sets of interacting proteins. Finding these interaction partners and mostly the interacting epitopes is crucial to the study of pathophysiological conditions and to the future of therapeutics. Because of the magnitude of such a task, several novel high throughput approaches have been developed including theoretical ab initio predictions, computer-aided in silico design, and large scale efforts based on various combinatorial chemistry strategies. Regardless of the approaches, recurrent problems in large scale methods often include their poor signal-to-noise ratio due to important numbers of false positive and negative hits, their lack of reproducibility, and the poor results obtained when searching for epitopes against membrane proteins (for a review, see Supplemental Discussion). More recently, efforts were centered on in vivo tagging systems coupled with mass spectroscopy and improved combinatorial display techniques (for reviews, see Refs. 1 and 2). The latter methodologies allow the selection of putatively relevant epitopes from a large molecular library by a screening process, and over the years, the yeast two-hybrid (3) and phage display (4) methods have been complemented by various types of cellular or ribosomal display with larger o...

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