RESUMO
Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be â¼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.
Assuntos
Escherichia coli K12/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fases de Leitura Aberta , Cromatografia Líquida de Alta Pressão , Escherichia coli/enzimologia , Escherichia coli/fisiologia , Escherichia coli K12/enzimologia , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Anotação de Sequência Molecular , Fosfogluconato Desidrogenase/química , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteogenômica/métodos , Proteômica/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas em TandemRESUMO
Recent advances in mass spectrometry-based proteomics have revealed translation of previously nonannotated microproteins from thousands of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes. Facile methods to determine cellular functions of these newly discovered microproteins are now needed. Here, we couple semiquantitative comparative proteomics with whole-genome database searching to identify two nonannotated, homologous cold shock-regulated microproteins in Escherichia coli K12 substr. MG1655, as well as two additional constitutively expressed microproteins. We apply molecular genetic approaches to confirm expression of these cold shock proteins (YmcF and YnfQ) at reduced temperatures and identify the noncanonical ATT start codons that initiate their translation. These proteins are conserved in related Gram-negative bacteria and are predicted to be structured, which, in combination with their cold shock upregulation, suggests that they are likely to have biological roles in the cell. These results reveal that previously unknown factors are involved in the response of E. coli to lowered temperatures and suggest that further nonannotated, stress-regulated E. coli microproteins may remain to be found. More broadly, comparative proteomics may enable discovery of regulated, and therefore potentially functional, products of smORF translation across many different organisms and conditions.
Assuntos
Proteínas e Peptídeos de Choque Frio/genética , Escherichia coli/genética , Proteínas/genética , Proteômica , Proteínas e Peptídeos de Choque Frio/isolamento & purificação , Anotação de Sequência Molecular/métodos , Proteínas/isolamento & purificaçãoRESUMO
Here we describe a new BODIPY-based membrane probe (1) that provides an alternative to dialkylcarbocyanine dyes, such as DiI-C18, that can be excited in the blue spectral region. Compound 1 has unbranched octadecyl chains at the 3,5-positions and a meso-amino function. In organic solvents, the absorption and emission maxima of 1 are determined mainly by solvent acidity and dipolarity. The fluorescence quantum yield is high and reaches 0.93 in 2-propanol. The fluorescence decays are well fitted with a single-exponential in pure solvents and in small and giant unilamellar vesicles (GUV) with a lifetime of ca. 4 ns. Probe 1 partitions in the same lipid phase as DiI-C18(5) for lipid mixtures containing sphingomyelin and for binary mixtures of dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC). The lipid phase has no effect on the fluorescence lifetime but influences the fluorescence anisotropy. The translational diffusion coefficients of 1 in GUVs and OLN-93 cells are of the same order as those reported for DiI-C18. The directions of the absorption and emission transition dipole moments of 1 are calculated to be parallel. This is reflected in the high steady-state fluorescence anisotropy of 1 in high ordered lipid phases. Molecular dynamic simulations of 1 in a model of the DOPC bilayer indicate that the average angle of the transition moments with respect to membrane normal is ca. 70°, which is comparable with the value reported for DiI-C18.
Assuntos
Alcanossulfonatos/química , Compostos de Boro/química , Membrana Celular/química , Corantes Fluorescentes/química , Lipossomas Unilamelares/química , Animais , Linhagem Celular , Polarização de Fluorescência , Simulação de Dinâmica Molecular , Ratos , Espectrometria de FluorescênciaRESUMO
The UV-vis electronic absorption and fluorescence emission properties of 8-halogenated (Cl, Br, I) difluoroboron dipyrrin (or 8-haloBODIPY) dyes and their 8-(C, N, O, S) substituted analogues are reported. The nature of the meso-substituent has a significant influence on the spectral band positions, the fluorescence quantum yields, and lifetimes. As a function of the solvent, the spectral maxima of all the investigated dyes are located within a limited wavelength range. The spectra of 8-haloBODIPYs display the narrow absorption and fluorescence emission bands and the generally quite small Stokes shifts characteristic of classic difluoroboron dipyrrins. Conversely, fluorophores with 8-phenylamino (7), 8-benzylamino (8), 8-methoxy (9), and 8-phenoxy (10) groups emit in the blue range of the visible spectrum and generally have larger Stokes shifts than common BODIPYs, whereas 8-(2-phenylethynyl)BODIPY (6) has red-shifted spectra compared to ordinary BODIPY dyes. Fluorescence lifetimes for 6, 8, and 10 have been measured for a large set of solvents and the solvent effect on their absorption and emission maxima has been analyzed using the generalized Catalán solvent scales. Restricted rotation about the C8-N bond in 7 and 8 has been observed via temperature dependent (1)H NMR spectroscopy, whereas for 10 the rotation about the C8-O bond is not hindered. The crystal structure of 8 demonstrates that the short C8-N bond has a significant double character and that this N atom exhibits a trigonal planar geometry. The crystal structure of 10 shows a short C8-O bond and an intramolecular C-H···π interaction. Quantum-chemical calculations have been performed to assess the effect of the meso-substituent on the spectroscopic properties.
RESUMO
8-Halogenated boradiaza-s-indacenes can be efficiently prepared from dipyrrylketones. The new dyes react smoothly with nucleophiles to yield N-, O-, and S-substituted chromophores, as well as transition-metal-catalyzed cross-coupling reactions. The nature of the new substitutent has a strong influence on the spectral properties of the dyes.