A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

Abstract

BackgroundClostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology.ResultsA two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation.ConclusionTwo dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.

Mono-dimensional blue native-PAGE and bi-dimensional blue native/urea-PAGE or/SDS-PAGE combined with nLC–ESI-LIT-MS/MS unveil membrane protein heteromeric and homomeric complexes in Streptococcus thermophilus

“…The second (at 371 kDa) and the third one (at 333 kDa) showed progressive disappearance of , or and subunits, respectively, which was associated with a partial F 1 complex decomposition. Our results were in good agreement with previous observations on other G+ and Gbacteria [40,[43][44][45][46][47]52,53].…”

“…Generally, their molecular mass value was in good agreement with that expected theoretically. Exceptions regarded poorly-represented epsB-epsC-containing complexes at 602 and 518 kDa, for which the occurrence of additional constituents (in low amounts) escaping a positive MS identification may be hypothesized, as already reported for other poorly-abundant protein complexes from other bacteria [44,45,52,55,57].…”

“…Alternatively, each protein complex band from 1D-BN-PAGE can be directly analyzed for its constituents by nLC-ESI-MS/MS [41]. Both approaches were used for high-throughput characterization of: i) membrane protein complexes from Neisseria meningitides [42], Rhodobacter sphaeroides [43], Francisella tularensis [44], Clostridium thermocellum [45], Mycobacterium bovis [46] and Enterococcus faecalis [47]; ii) cytoplasmic protein complexes from Streptomyces coelicolor [48] and Pseudomonas sp. [49]; iii) membrane and cytoplasmic protein complexes from…”

A comparative proteomic analysis of the early response to compatible symbiotic bacteria in the roots of a supernodulating soybean variety

“…Among the bacterial proteins, glycosyltransferase, TetR family transcriptional regulator, and ATP synthase were upregulated, while translational elongation factor Tu, SOS response transcriptional repressor, and AAA ATPase were downregulated during the early stages of the symbiotic interaction between B. japonicum and the normal-nodulating Enrei variety. F0F1 ATP synthase, a subtype of prokaryotic ATPase/synthase [30] and key enzyme in energy metabolism, was upregulated during the symbiotic interaction. Exopolysaccharides act as signals to plant hosts to initiate infection thread formation [31], and their biosynthesis requires glycosyltransferases, an enzyme that transfers nucleotide diphospho-sugars to growing polysaccharide chains [32].…”

Proteomic identification of hair cell repair proteins in the model sea anemone Nematostella vectensis

“…Blue native PAGE was employed to compare native protein complexes occurring in control mucus to those found in trauma mucus. Blue native PAGE was developed to maintain the native state of proteins and protein complexes during separation (Sch€ agger and von Jagow, 1991) and has been used in many studies, including those aimed at isolating mitochondrial and membrane protein complexes (reviewed in Nijtmans et al, 2002;Peng et al, 2011). Because control mucus lacks repair activity, key RPs were hypothesized to be enriched in trauma mucus, or exclusively present in trauma mucus.…”