Mass estimation of native proteins by blue native electrophoresis: principles and practical hints.

Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results. We present short protocols taking 10-30 min to prepare optimal sets of membrane protein markers from chicken, rat, mouse, and bovine heart. Especially heart materials from local supermarkets or butcher's shops, e.g. chicken or bovine heart, are ideal sources of high mass membrane protein standards. Considerable discrepancies between the migration behavior of membrane and soluble markers suggest using membrane protein markers for mass estimation of membrane proteins. Soluble standard proteins can be used, with some limitations, when soluble proteins are the focus. Principles and general rules for the determination of mass and oligomeric state of native membrane and soluble proteins are elaborated, and potential pitfalls are discussed.

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.

Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: isolation of putative respiratory strings or patches.

Here, we expand the application of blue native electrophoresis to the separation of mega protein complexes larger than 10 MDa by introducing novel large pore acrylamide gels. We tailored the bis-acrylamide cross-linker amounts relative to the acrylamide monomer to enlarge the pore size of acrylamide gels and to obtain elastic and sufficiently stable gels. The novel gel types were then used to search for suprastructures of mitochondrial respiratory supercomplexes, the hypothetical respiratory strings, or patches. We identified 4-8 MDa assemblies that contain respiratory complexes I, III, and IV and most likely represent dimers, trimers, and tetramers of respiratory supercomplexes. We also isolated multimeric respiratory supercomplexes with apparent masses of 35-45 MDa, the presumed core pieces of respiratory strings or patches. Electron microscopic investigations will be required to clarify whether the isolated assemblies of complexes are ordered and specific, as predicted for respiratory strings and patches in the mitochondrial membrane.

Native immunoblotting of blue native gels to identify conformation-specific antibodies.

A large repertoire of immunological methods permits monitoring the interaction of antibodies with their specific antigen. However, recognition of a protein by a conformation-specific antibody represents a challenge because native conditions must be kept throughout the assay. Native immunoblotting of blue native gels conserves the native state by using Tween 20 instead of methanol for the obligatory destaining of the blot membrane. We validate the new technique with a set of monoclonal antibodies against respiratory NADH:ubiquinone oxidoreductase.

Chapter 8 Two-dimensional native electrophoresis for fluorescent and functional assays of mitochondrial complexes.

Supramolecular assemblies of native membrane protein complexes were solubilized from biological membranes by very mild detergents and isolated by native electrophoresis. The complexity of these higher order structures can be reduced for proteomic investigations by applying less mild native electrophoresis variants in the second dimension. Supercomplexes thereby dissociate into the individual complexes. Clear-native and blue-native electrophoresis variants are useful alternatives for the second native dimension, but clear-native electrophoresis is advantageous for the identification of fluorescence-labeled proteins and for in-gel activity assays that are hampered by Coomassie dye. The 2-D native gels comprising two orthogonal native dimensions are useful to determine the stoichiometry of complexes in supercomplexes. Strips from 2-D native gels can also be used for 3-D SDS-PAGE to identify loosely bound accessory subunits of supercomplexes. The subunit composition of supercomplexes and individual complexes is investigated by 4-D gels. The 4-D protocol starts with isolation of highly pure membrane protein complexes by 2-D native electrophoresis, followed by doubled SDS-PAGE to resolve the subunits.