Hunting interactomes of a membrane protein: obtaining the largest set of voltage-dependent anion channel-interacting protein epitopes.
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.
Roman
Inge
I
Department of Physiology (FYSP), Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium.
Figys
Jurgen
J
Steurs
Griet
G
Zizi
Martin
M
D001435:Bacteriophages; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D008081:Liposomes; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D050994:Voltage-Dependent Anion Channels / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D008081:Liposomes; D008565:Membrane Proteins; D019151:Peptide Library; D050994:Voltage-Dependent Anion Channels