| Abstract |
In the present study, the expression and the topology of the proteins of Chl. tepidum were examined, by combining proteomics fractionation techniques with gel-free proteomics analysis (N-terminal COFRADIC and nLC-MS/MS). Additionally, isolated chlorosomes from Chl. tepidum were studied, regarding their size, using DLS and EM, and their protein composition, using nLC-MS/MS.
Chl. tepidum is a thermophile, Gram-negative green-sulfur bacterium. It is an obligatory anaerobic photolithoautotroph, found in aquatic environments where the higly abundant in reduced sulfur compounds anoxic layers are exposed to light. Green sulfur bacteria use a unique apparatus for light harvesting, the chlorosome, in which pigments are organised in aggregates, rather than associated with proteins.
Chl. tepidum is a model organism for the study of inorganic sulfur metabolism and photosynthesis. The genome of chl. Tepidum has recently been sequenced and it was found to comprise of 2288 assigned coding sequences. The function of a large number of encoded proteins remains unknown, however many of these proteins appear to be highly conserved among photosynthetic organisms. Therefore, it can be inferred that these proteins may be functionally involved in photosynthesis. In addition, although studies based on SDS-PAGE and immunodetection have astablished that chlorosomes contain 10 gene products, their function, as well as the protein composition of intact chlorosomes is yet to be determined.
The analysis and characterization of the gene products of a cell, tissue or organism is termed proteomics. Proteomic analysis is a very interesting research field, despite its limitiations regarding the increased sample complexity, their diverse physical and chemical properties and the dynamic range of the protein concnetrations in any given proteome. The reason why this field attracts so much academic interest is because it is proteins which are responsible for the phenotype of the cells and because the cells’ functions cannot be fully understood only by genomic studies.
In this study, the proteome of Chl. tepidum was fractionated, and the resulting cytoplasmic, periplasmic, total membrane and water-soluble sub-proteomes were characterized using absorption spectroscopy and Tricine SDS-PAGE, and they were subsequently analysed using N-terminal COFRADIC. N-terminal COFRADIC is an analytical technique, aiming through chemical modifications and chromatography techniques, to select the N-terminal peptides that emerge after trypsin digestion of the protein sample. After the selection of this class of peptides, the less complex peptide mixture is subjected to nLC-MS/MS analysis.
The N-terminal COFRADIC analysis of the fractionated proteome of Chl. tepidum, led to the identification of 805 proteins, most of which, based on their predicted subcellular topology [51-52] are cytoplasmic proteins, whereas 277 were of unknown topology. With regards to their annotated function [3], the majority of the identified proteins were of unknown function, whereas most of the remaining proteins are involved in energy metabolism. The identification of a large number of proteins in some protein fractions but not in all of them, stresses the importance of prefractionation techniques in proteomic analysis, as well as the importance of selecting a representative class of peptides, such as N-terminal peptides, in order to identify the largest possible number of proteins in such complex protein samples.
Furthermore, the chlorosome fraction of Chl. tepidum was isolated and characterized using absorption spectroscopy, Tricine SDSPAGE, electron microscopy and dynamic light scattering (DLS). Tricine SDS-PAGE showed the presence of at least twenty proteins with molecular weights ranging between 6 kDa and 70 kDa. Light scattering and electron microscopy revealed particles of 140 nm and 170 nm in length respectively. Additionally, the protein composition of the intact chlorosome was obtained by MS/MS-driven proteomics, leading to the identification of 270 proteins, including all 10 chlorosomal proteins, along with a number of proteins which are suggested to interact with the chlorosome [8, 12, 14].
All in all, the proteomic analysis of Chlorobium tepidum led to the identification of 891 proteins, accounting for 38.9% of the predicted proteome of Chl. tepidum [3]. Therefore, this proteomics study, has led to the identification of the largest number of proteins of Chlorobium tepidum to date, providing information about their state and topology.
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