title: A comparative cross-linking strategy to probe conformational changes in protein complexes.
abstract: Chemical cross-linking, together with mass spectrometry (MS), is a powerful combination for probing subunit interactions within static protein assemblies. To probe conformational changes in response to stimuli, we have developed a comparative cross-linking strategy, using lysine-specific deuterated and nondeuterated bis(sulfosuccinimidyl)suberate cross-linking reagents (BS3). Here we describe the experimental procedures as well as the data analysis, validation and interpretation. The protocol involves first assigning cross-linked peptides in the complex without ligand binding, or with post-translational modifications (PTMs) at natural abundance, using a standard procedure with labeled cross-linkers, proteolysis and assignment of cross-linked peptides after liquid chromatography-tandem MS (LC-MS/MS) and database searching. An aliquot of the protein complex is then exposed to a stimulus: either ligand binding or incubation with a phosphatase or kinase to bring about changes in PTMs. Two solutions--one containing the apo/untreated complex and the other containing the enzymatically modified/ligand-bound complex--are then cross-linked independently. Typically, nondeuterated BS3-d0 is used for the untreated complex and deuterated BS3-d4 is used for the experiment. The two aliquots are then incubated at equal concentrations, digested and processed as before. The ratios of labeled and unlabeled cross-linked peptides provide a direct readout of the effect of the stimulus. We exemplify our method by quantifying changes in subunit interactions induced by dephosphorylation of an ATP synthase. The protocol can also be used to determine the conformational changes in protein complexes induced by various stimuli including ligand/drug binding, oligomerization and other PTMs. Application of the established protocol takes ~9 d, including protein complex purification.
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK.
authors.forename: Carla
authors.initials: C
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008239:Lysine; D046912:Multiprotein Complexes / Q000737:chemistry*; D011487:Protein Conformation; D013388:Succinimides; D053719:Tandem Mass Spectrometry
chemical_list: D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D013388:Succinimides; C035760:bis(sulfosuccinimidyl)suberate; D008239:Lysine