Membrane Association of a Protein Increases the Rate, Extent, and Specificity of Chemical Cross-Linking

Abstract

Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used crosslinking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical crosslinking reactions with the trifunctional maleimide TMEA. CF binding to vesicles is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so crosslinking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Crosslinking specificity, which is measured as the preference for crosslinking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein-protein interactions, an important consideration for understanding processes involving membrane-associated proteins. The experiments further demonstrate the importance of crosslinking conditions for these reactions that are often used to probe protein structure and dynamics, and the potential of membrane association to restore native interactions of membrane-associated proteins for crosslinking studies.

Hydrogen exchange of chemoreceptors in functional complexes suggests protein stabilization mediates long-range allosteric coupling

“…This suggests that MH2/MH2' forms the dimer interface, since MH1 shows no evidence of stable packing (log(PF) ≤ 2.4). Such a model is consistent with a recent mutagenesis study suggesting stable MH2/MH2' packing at the membrane proximal end of MH2 in the Ser receptor (25), but is difficult, however, to reconcile with the existence of disulfide crosslinked MH1 /MH1' dimers within this region that retain activity (5,26).…”

Cysteine Residues Impact the Stability and Micelle Interaction Dynamics of the Human Mitochondrial β-Barrel Anion Channel hVDAC-2

“…While mass spectrometric analysis has established that disulfides are largely absent in refolded hVDAC-2, disulfide-mediated oligomerization may be induced as a consequence of changes in LPR. However, we observe comparable oligomerization (which together constitute ∼40% of the total protein; ∼60% stays monomeric) in both WT and C0 barrels in all LPRs, in formaldehyde-mediated cross-linking experiments(Figure S4 in File S1 ).However, we do not detect defined dimeric and trimeric forms observed previously for hVDAC-1 [48] and rat liver mitochondrial VDAC [49] , which could be attributed either to the tendency of hVDAC-2 to form multimeric structures, or to non-specific cross linking by formaldehyde.The presence of a two-fold excess DTT (the default concentration is 2 mM DTT) in WT samples showed an additional increase in the cross-linked species (∼55% oligomers), that can arise due to the increase in cross-linking efficiency upon membrane association of Cys-containing proteins, as observed earlier [50] . The oligomerization was more profound when other cross-linking agents such as glutaraldehyde or EGS were used in place of formaldehyde (data not shown).…”

New Insights into Bacterial Chemoreceptor Array Structure and Assembly from Electron Cryotomography

“…All lipids were purchased from Avanti Polar Lipids, and large unilamellar vesicles (LUVs) were prepared as previously described. 31 PEG 8000 (Fluka) and d -(+)-trehalose (Sigma-Aldrich) were prepared as 40% (w/v) stock solutions in deionized water and passed through a 0.22 μm syringe filter prior to being used. A modified kinase buffer [50 mM potassium phosphate, 50 mM KCl, and 5 mM MgCl 2 (pH 7.5)] was used for sample preparation.…”

“…Formation and characterization of kinase-active ternary complexes followed published methods, 30 , 31 further specified as follows. Vesicle-mediated CF 4Q ternary complexes were prepared by incubating 30 μM CF, 12 μM CheW, and 6 μM CheA with 580 μM total LUVs (1:1 DOPC:DOGS-NTA-Ni 2+ ratio), while PEG-mediated CF 4Q complexes were prepared by incubating 50 μM CF, 20 μM CheW, and 12 μM CheA with final concentrations of 7.5% (w/v) PEG 8000 and 4% (w/v) trehalose.…”