Dataset Information

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

ABSTRACT: We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

SUBMITTER: Barrera NP

PROVIDER: S-EPMC4066579 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

Barrera Nelson P NP Isaacson Shoshanna C SC Zhou Min M Bavro Vassiliy N VN Welch Alex A Schaedler Theresia A TA Seeger Markus A MA Miguel Ricardo Núñez RN Korkhov Vladimir M VM van Veen Hendrik W HW Venter Henrietta H Walmsley Adrian R AR Tate Christopher G CG Robinson Carol V CV

Nature methods 20090705 8

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes. ...[more]

PMID: 19578383

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Project description:Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (?, ?, ?, and ?). The catalytic protein kinase subunit within this complex is ?, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the ? subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple ? subunits, arguing for their self-association within the activated complex. Evidence for the theoretical ???? protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.

Dataset Information

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

Publications

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

Similar Datasets

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