| Abstract |
Chlorobium tepidum is a Gram-negative, thermophilic, green sulfur bacterium (Chlorobia), obligate anaerobic photolithoautotroph and is widely distributed in aquatic environments, where anoxic layers containing reduced sulfur compounds are exposed to light. Recently, the genome from the green sulfur bacterium Chl. tepidum was sequenced but the function of a large number of genes remains unknown. Many genes were found to be highly conserved among photosynthetic species and seemed to have no clear function within Chl. tepidum, however, these genes are thought to play specific roles in photosynthesis or photobiology. Characterization of different gene products can be achieved through proteomics approaches. The corresponding genes may then be identified from the genome sequence by determining the N-terminal sequence of the protein or by using mass spectroscopic sequencing and identification methods. An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including membrane proteins, which are rarely detectable on two-dimensional electrophoresis gels. In this study, different methods were employed for the proteomic analysis of the soluble and membrane fraction of Chlorobium tepidum. Initially, we tried the enrichment of membrane proteins from Chlorobium tepidum prior to analysis with two-dimensional electrophoresis (2-DE). Isolated membranes were solubilized with Triton X-100 and from the supernatant we identified 58 unique proteins. The use of ionic sodium dodecyl sulfate (SDS) for protein solubilization, combined with acetone precipitation, resulted in an improved 2-DE pattern and the total number of the identified proteins was increased to 117. The use of acetone for protein precipitation improved the results by extracting compounds potentially deleterious to the resolution of 2-DE. However, the additional proteins detected by the use of SDS are in themajoritymore difficult to solubilize than less hydrophobic proteins. Further our attempts for selective extraction of the outer membrane proteins using the acid glycine method allowed the identification of 37 proteins of which 14 were predicted to have a signal sequence indicating their localization in the periplasmic space or in the outer membrane. The identification of a significant amount of soluble proteins, especial ribosomal proteins, in the membrane fraction of Chl. tepidum indicated the necessity to wash the membranes prior any further analysis. For that purpose the isolated membranes of Chl. tepidum were washed with two different salts, EDTA and NaBr. The washed membranes in both procedures were collected with ultracentrifugation. The supernatant and the membranes resulted from both procedures were analyzed with absorption spectroscopy, Tricine-SDS-PAGE and the total amount of protein was estimated using the Bradford assay. Tricine-SDS-PAGE and Bradford assay showed that the washed with EDTA membranes contained more proteins than the washed with NaBr membranes. In total, using MALDI TOF MS 119 different proteins were identified from the Tricine-SDS-PAGE of the washed with EDTA membranes, while only 93 different proteins were identified from the Tricine-SDS-PAGE of the washed with NaBr membranes. The supernatant from the two different procedures were different. In the case of the washed with EDTA membranes the supernatant was transparent having a light redbrown color, while the supernatant in the case of NaBr was separated in two distinct phases. One viscous dark green phase and one transparent light olive green phase. Both phases showed a relatively high absorbance at 750 nm indicating the presence of BChlc therefore the presence of chlorosomes in the supernatant. On the contrary, the supernatant from the washed with EDTA membranes showed only characteristic peaks of carotenoids and cytochrome type c. The quality of the Tricine-SDS-PAGE of the supernatant from the procedure with EDTA was better regarding the resolution and the number of the separated protein bands. In addition, the total number of the identified proteins from the Tricine- SDS-PAGE of the supernatant from the procedure with EDTA was 106, while from the Tricine-SDSPAGE of the supernatant from the procedure with NaBr were 60. The vast majority of the identified proteins were soluble and mainly ribosomal proteins. In the case of NaBr we noticed the removal of several peripheral membrane proteins from their membrane complexes (like PscD from the RC) which will probably cause the instability or the inactivation of the membrane complexes. This is not preferable in a functional study of the membrane proteins. Another bottleneck for the proteomic analysis of the membranes of Chl. tepidum is the presence of chlorosomes which contain a huge amount of pigments that influence any electrophoretic analysis. We tried to isolate chlorosome-depleted membranes for the analysis of the membrane proteome of Chl. tepidum. The membranes were purified by sucrose density centrifugation and characterized by one-and two-dimensional electrophoresis (1-DE, 2-DE) coupled with mass spectrometry, absorption spectroscopy and electron microscopy. 1-DE and 2-DE were employed to analyze the membrane proteins and to characterize the capabilities of the methods. At the same time, improvement of the solubilization of the membrane proteins prior to 2-DE was accomplished using a series of zwitterionic detergents. Based on the resolved spots after 2-DE, the combination of ASB-14 with Triton X-100 is more efficient than the combination of CHAPS, SB 3-10 and Triton X-100. From the application of 1- DE and 2-DE, 167 and 202 unique proteins were identified respectively, using peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Both methods resulted in the detection of 291 different proteins of which only 88 were predicted membrane proteins, indicating the limitation of membrane proteins detection after separation with electrophoresis methods. In addition 53 of them were identified as outer membrane proteins indicating the close association of outer membrane proteins to the plasma membrane. We investigated protein-protein interactions in the membrane and soluble fraction of Chl. tepidum utilizing blue-native polyacrylamide gel electrophoresis (BN-PAGE) in combination with 2-D BN/Tricine-SDSPAGE and identification of the separated proteins by mass spectrometry. The soluble proteins and the chlorosomes were removed combining the treatment of the isolated membranes from Chl. tepidum with EDTA and NaBr, and the ultracentrifugation on sucrose gradient of the washed membranes, prior the functional study. Three major bands were separated, which were studied with visible spectroscopy and Tricine-SDS-PAGE. Chlorosomes were isolated in one band close to the top of the sucrose gradient, while the lower bands close to the bottom of sucrose gradient, which showed the lowest absorbance at 750 nm and the highest at 810 nm, was analyzed with ΒΝ-PAGE and 2-D BN/Tricine-SDSPAGE. Comparing the ΒΝ-PAGE gels resulted from the different washing procedures of the membranes, was ascertain that except the different number of protein complexes (18 in NaBr and 17 in EDTA) the main difference is the existence of a wide protein band in the middle of the gel, with an apparent molecular weight of 232 kDa. This protein band consists mainly of the hypothetical protein Q8KBI3, which has been identified in every of our studies on the membranes of Chl. tepidum, and possess characteristics of an outer membrane protein. It seems to incur in at least two isoform, which form protein complexes with different size. In total, 33 different proteins were identified after ΒΝ-PAGE separation and MALDI TOF MS analysis. The majority of those proteins is membrane and is related with the energy metabolism. The GRAVY values of the majority of the proteins are similar with hydrophilic proteins with none or small hydrophobic regions in their amino acid sequence. From 2-D BN/Tricine-SDSPAGE around 100 spots resulted from which 89 identified which corresponds to 120 different proteins. The GRAVY values of the majority of the proteins correspond to more hydrophobic proteins in regard with the proteins which were identified in 1-D BN-PAGE. The big subunit of RC of Chl. tepidum was identified in 8 protein complexes on the top of ΒΝ-PAGE gel. In 7 of them FMO protein was also identified. The apparent molecular weight of those complexes is in agreement with the calculated molecular weight of the RC complex (581 kDa). The subunits of RC, PscΒ, PscC and PscD were not detected in any complex in ΒΝ-PAGE, but the apparent molecular weight of the complexes in which PscA was identified, indicates the existence of the whole complex of RC. FMO protein seems to occur in different complexes with RC, either as two trimers or as one, and as one free trimer without the RC The chlorosome-depleted membranes which resulted from sucrose gradient of the washed membranes were solubilized with DDM and analyzed with 1-D BN-PAGE. 24 protein complexes resulted in all three bands, from which 18 different proteins were identified. The GRAVY values of the majority of the proteins are similar with hydrophilic proteins with none or small hydrophobic regions in their amino acid sequence. From 2-D BN/Tricine-SDS-PAGE, 47 protein spots resulted from which 25 different proteins were identified by MALDI TOF MS. The GRAVY values of the majority of the proteins are similar with hydrophilic proteins. The subunits of the photosynthetic RC, PscA and PscC were identified in the 1-D gel but not in the 2-D. The RC of Chl. tepidum occurs solubilized in different intermediate complexes with apparent molecular weights between 400 and 800 kDa, which is in agreement with the bibliography. 2-D BN/Tricine-SDS-PAGE confirmed the ascertainment from 1-D ΒΝ-PAGE, that the hypothetical proteins Q8KBI3 and Q8KE01 form a membrane complex. These proteins have similar molecular weight which makes their separation in the 2-D not possible. BN-PAGE of the soluble proteins of Chl. tepidum resulted the separation of 42 protein complexes from which 62 different proteins were identified by MALDI TOF MS. The combination of 1-D BN-PAGE with 2-D ΒΝ/Tricine-SDS PAGE obtained the separation of more than 200 protein spots out of which 188 different proteins were identified. 141 (75%) proteins of them were not identified in the 1-D BN-PAGE, a fact that indicates the necessity of 2-D ΒΝ/Tricine-SDS PAGE for the identification of all the proteins that participate in one protein complexes. Characteristic cytosolic protein complexes such as RNA polymerase and chaperonin were identified. In addition, proteins which have important roles in metabolism and in particular sulfur metabolism were identified significant evidences came up for their interaction with specific proteins. Globally, from the proteomic analysis that we realized in the Chl. tepidum identified in total 602 proteins from the 2,288 proteins that are predicted by the genome. This number constitutes the 26.31% of the theoretical proteome of Chl. tepidum. This is the first time that 109 hypothetical proteins are experimentally identified, where the 39 from which they are conserved between various photosynthetic organisms and likely they play important role in the photosynthesis and more generally in photobiology.
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Πρωτεομική ανάλυση του πράσινου θειοβακτήριου Chlorobium tepidum
