Addressing Shewanella oneidensis "cytochromome": the first step towards high-throughput expression of cytochromes c

Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain-are essential in most organisms. However, they are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. We have addressed this challenge by designing two families of vectors (total of 6 vectors) suitable for ligation-independent cloning and developing a pipeline for expression and solubility analysis of cytochromes c. This system has been validated by expression analysis of thirty genes from Shewanella oneidensis coding for cytochromes c or cytochromes c-type domains predicted to have 1-4 hemes. Out of 30 targets, 26 (87%) were obtained in soluble form in one or more vectors. This work establishes a methodology for high-throughput expression of this class of proteins and provides a clone resource for the microbiological and functional genomics research communities.     

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of d-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains l-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.     

Design and initial characterization of the SC-200 proteomics standard mixture

High-throughput (HTP) proteomics studies generate large amounts of data. Interpretation of these data requires effective approaches to distinguish noise from biological signal, particularly as instrument and computational capacity increase and studies become more complex. Resolving this issue requires validated and reproducible methods and models, which in turn requires complex experimental and computational standards. The absence of appropriate standards and data sets for validating experimental and computational workflows hinders the development of HTP proteomics methods. Most protein standards are simple mixtures of proteins or peptides, or undercharacterized reference standards in which the identity and concentration of the constituent proteins is unknown. The Seattle Children's 200 (SC-200) proposed proteomics standard mixture is the next step toward developing realistic, fully characterized HTP proteomics standards. The SC-200 exhibits a unique modular design to extend its functionality, and consists of 200 proteins of known identities and molar concentrations from 6 microbial genomes, distributed into 10 molar concentration tiers spanning a 1,000-fold range. We describe the SC-200's design, potential uses, and initial characterization. We identified 84% of SC-200 proteins with an LTQ-Orbitrap and 65% with an LTQ-Velos (false discovery rate?=?1% for both). There were obvious trends in success rate, sequence coverage, and spectral counts with protein concentration; however, protein identification, sequence coverage, and spectral counts vary greatly within concentration levels.     

A human histone H2B.1 variant gene, located on chromosome 1, utilizes alternative 3' end processing.

A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.