Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins <em>In Vivo</em>

Abstract

Membrane proteins are essential for cell viability and are therefore important therapeutic targets [1][2][3] . Since they function in complexes 4 , methods to identify and characterize their interactions are necessary 5. To this end, we developed the Membrane Strep-protein interaction experiment, called . This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins. Video LinkThe video component of this article can be found at

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC

“…The frozen pellets were subjected to membrane isolation based on published procedures ( 39 , 50 ) with some modifications. Briefly, cell pellets were suspended in 8-ml portions of buffer A (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA) supplemented with 10 mM MgCl 2 , 10 U/ml DNase and RNase, and 0.1 ml of 1 M phenylmethylsulfonyl fluoride (PMSF).…”

The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli

“…2b ). The Membrane-SPINE approach combines the purification of a specific Strep-tagged membrane protein with the reversible fixation of protein complexes by adding the aforementioned cross linker formaldehyde, allowing snapshots of interactions in living cells [ 48 ]. Here we used a MG1655-derivate E. coli strain harboring a chromosomal fusion of the arcA gene with a Snap-Tag and a fusion of cpxA with a Strep-Tag on the medium copy plasmid pMal-p2X (~20 copies per cell; [ 49 ]).…”

“…Membrane-SPINE was performed as described previously with min minor modifications [ 48 ]. In brief, GP01E cells (chromosomal fusion of arcA with Snap-tag in MG1655) were transformed with pKT01E harboring a C-terminal fusion of cpxA with Strep-tag.…”

Formaldehyde cross-linking and structural proteomics: Bridging the gap

“…Following enzymatic digestion, LC-MS/MS analyses identifies proteins via the large abundance of unmodified peptides present [34]. This has been successful in determining protein interacting partners in various species [3,4,34,[38][39][40][41][42][43][44][45][46][47][48][49][50]. For example, formaldehyde's capacity to quickly diffuse through and freeze protein geometries in living biological material has triggered large-scale cross-linking of even whole organisms through time-controlled transcardiac perfusion cross-linking (tcTPC) [40,42,45].…”