Novel Multiprotein Complexes Identified in the Hyperthermophilic Archaeon Pyrococcus furiosus by Non-denaturing Fractionation of the Native Proteome

Abstract

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing ϳ80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.

Synthesis, characterization and DNA-binding properties of Ru(II) complexes coordinated by ofloxacin as potential antitumor agents

“…The titration processes were repeated until the spectra did not change for at least four titrations, indicating that binding saturation was achieved. The intrinsic binding constants [20] for the complexes with CT DNA at intraligand (IL) band were calculated following equation (1) [21] and according to the decay of IL band in the presence of DNA [19], . These results may be attributed to the structural differences between three complexes which have different bipyridine or phenanthroline coordinated to Ru.…”

Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome

“…Cytoplasmic extracts of each strain were prepared as previously described (42). All steps were carried out under strict anaerobic conditions unless otherwise noted.…”

“…This organism has been studied by numerous "-omics"-based approaches, including transcriptomics by tiling (59) and DNA microarrays (14,35,51,54,57), comparative genomics (8,33,60), proteomics (32,42), metallomics (11), and structural genomics (25). In many ways, it has become one of the model hyperthermophiles, a status recently sustained by the development of a genetic system for the organism (37).…”

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

“…While we failed to identify the Cst1 subunit by our immunopurification approach (Fig. 2), Pfu Cst1 was found in complex with both Cst2 and Cas5t following sequential nondenaturing column chromatography fractionations (Menon et al 2009). Collectively, the data suggest that all three Cst proteins (Cst1, Cst2, Cas5t) are integral subunits of Type I-G crRNPs.…”