Nearly one-third of all genes in various organisms encode membrane associated proteins that participate in numerous protein-protein interactions important to the processes of life. Various methods have been developed to identify and characterize these interactions including pull-down, yeast two-hybrid and colocalization methods, among others. However, membrane protein complexes pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Aiming at development of protocols for the identification and characterization of membrane protein interactions, we have tested the feasibility of isolating and identifying the several protein complexes in Rhodopseudomonas palustris. R. palustris is a purple, non-sulfur phototrophic bacterium commonly existing in soils and water. Towards this purpose, subunits of several complexes were independently cloned with both a 6-histidine tag and a V5 epitope tag at the protein C-terminal end and used as baits for isolation of complexes using endogenous pull-down methods as well as protein correlation profiling chromatographic methods. Cells expressing individual tandem tag fused proteins were then cultured under illumination at 30oC under anaerobic conditions.

Cells solubilized with non-ionic or zwitterionic detergents were subjected to affinity “pull-down” experiments, using either Ni-NTA gels alone or a two-step tandem procedure, first with Ni-NTA, and then using a V5 epitope specific antibody. Tryptic digested peptide fragments of the pull-down products were then subjected to LC-MS/MS analysis. Results will be shown for the ATP synthase, photosynthetic reaction center and NADH dehydrogenase complexes.

In addition to endogenous pull-down experiments, protein complexes have been elucidated using protein correlation profiling completed employing tagged “bait” protein cell lines. Using this method, protein complexes are identified and characterized by matching elution profiles from multidimensional separation techniques. The keys to success of such an approach are that the combination of various separation strategies provides sufficient aggregate discrimination to achieve a fractionation pattern that defines a single complex.

To complete this method, cell lysate was first detergent solubilized and pre-fractionated using differential ammonium sulfate precipitation under gentle conditions to maintain intact protein complexes. Fractions containing the bait protein (as detected via Western blot assay) were each further separated using anion exchange chromatography. Assuming the complex is stable under these separation conditions, proteins that constitute a complex should co-fractionate. Complexes were identified by following the elution profile of the tagged protein via Western blot analysis in conjunction with LC-MS/MS of the various elution fractions. Further resolution of complexes was completed using Blue Native PAGE of elution fractions followed by in-gel digestion of bands and LC-MS/MS analysis. Using the tagged approach, multiple complexes may also be identified within the same anion exchange chromatography experiment.

Pull-down assays and protein correlation profiling may be used as orthogonal methods to characterize the same complex in order to increase the confidence of protein-protein interaction identifications. Results using these methods will be presented for several different protein complexes.

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