RESUMO
In this study, we performed the first large-scale identification of N-terminal peptides from the green sulfur bacterium Chlorobaculum tepidum. Combined fractional diagonal chromatography (COFRADIC) was used to isolate protein N-terminal peptides from three different proteome preparations, and following LC-MS/MS analysis, over 621 different proteins were identified by their N-terminal peptides. Our data constitute the largest data set currently available for protein N-termini of prokaryotic photosynthetic organisms.
Assuntos
Proteínas de Bactérias/metabolismo , Chlorobi/metabolismo , Fragmentos de Peptídeos/química , Proteoma/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cromatografia Líquida , Sequência Conservada , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Proteólise , Proteoma/química , Proteômica , Análise de Sequência de Proteína , Frações Subcelulares , Espectrometria de Massas em TandemRESUMO
Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.
Assuntos
Proteínas de Bactérias/química , Chlorobi/química , Chlorobi/citologia , Espectrometria de Massas/métodos , Organelas/química , Organelas/ultraestrutura , Proteômica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Luz , Microscopia Eletrônica/métodos , Dados de Sequência Molecular , Proteoma/análiseRESUMO
Chlorobium tepidum is a Gram-negative bacterium of the green sulfur phylum (Chlorobia). Chlorobia are obligate anaerobic photolithoautotrophs that are widely distributed in aquatic environments where anoxic layers containing reduced sulfur compounds are exposed to light. The envelope of C. tepidum is a complex organelle composed of the outer membrane, the periplasm-peptidoglycan layer, and the cytoplasmic membrane. In addition to the outer and plasma membranes, C. tepidum contains chlorosomes attached to the cytoplasmic side of the plasma membrane. Each cellular compartment has a unique set of proteins, called sub-proteome. An important aim of proteome analysis is to study the level of the expressed genes and their response to environmental changes. Membrane protein studies are of primary importance to understand how nutrients are transported inside the cell, how toxic molecules are exported, and the mechanisms of photosynthesis and energy metabolism.
