Dataset Information

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

ABSTRACT: Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

SUBMITTER: Walian PJ

PROVIDER: S-EPMC3516867 | biostudies-other | 2012 Dec

REPOSITORIES: biostudies-other

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High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Walian Peter J PJ Allen Simon S Shatsky Maxim M Zeng Lucy L Szakal Evelin D ED Liu Haichuan H Hall Steven C SC Fisher Susan J SJ Lam Bonita R BR Singer Mary E ME Geller Jil T JT Brenner Steven E SE Chandonia John-Marc JM Hazen Terry C TC Witkowska H Ewa HE Biggin Mark D MD Jap Bing K BK

Journal of proteome research 20121025 12

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodo ...[more]

PMID: 23098413

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Project description:Among bacterial cell envelopes, the Borrelia burgdorferi outer membrane (OM) is structurally unique in that the identities of many protein complexes remain unknown; however, their characterization is the first step toward our understanding of membrane protein interactions and potential functions. Here, we used two-dimensional blue native/SDS-PAGE/mass spectrometric analysis for a global characterization of protein-protein interactions as well as to identify protein complexes in OM vesicles isolated from multiple infectious sensu stricto isolates of B. burgdorferi. Although we uncovered the existence of at least 10 distinct OM complexes harboring several unique subunits, the complexome is dominated by the frequent occurrence of a limited diversity of membrane proteins, most notably P13, outer surface protein (Osp) A, -B, -C, and -D and Lp6.6. The occurrence of these complexes and specificity of subunit interaction were further supported by independent two-dimensional immunoblotting and coimmunoprecipitation assays as well as by mutagenesis studies, where targeted depletion of a subunit member (P66) selectively abolished a specific complex. Although a comparable profile of the OM complexome was detected in two major infectious isolates, such as B31 and 297, certain complexes are likely to occur in an isolate-specific manner. Further assessment of protein complexes in multiple Osp-deficient isolates showed loss of several protein complexes but revealed the existence of additional complex/subunits that are undetectable in wild-type cells. Together, these observations uncovered borrelial antigens involved in membrane protein interactions. The study also suggests that the assembly process of OM complexes is specific and that the core or stabilizing subunits vary between complexes. Further characterization of these protein complexes including elucidation of their biological significance may shed new light on the mechanism of pathogen persistence and the development of preventative measures against the infection.

Project description:Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a ?-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes.IMPORTANCE Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core ?-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.

Dataset Information

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Publications

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

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