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Cross-linking Experiments (cross-linking + experiment)
Selected AbstractsDefective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coliFEBS JOURNAL, Issue 22 2002Hendrik Adams In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564,5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position ,10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon. [source] Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coliGENES TO CELLS, Issue 12 2004Toh-ru Shimuta Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 °C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ, a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor ,32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ. Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ -independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins. [source] A T3 allele in the CFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with Congenital Bilateral Absence of Vas Deferens (CBAVD),HUMAN MUTATION, Issue 1 2005Antoine Disset Abstract The different alleles at the (TG)m(T)n polymorphic loci at the 3, end of the human CFTR intron 8 determine the efficiency by which exon 9 is spliced. We identified a novel TG12T3 allele in a congenital bilateral absence of vas deferens (CBAVD) patient who carries a [TG11T7; p.Phe508Cys; p.Met470Val] haplotype on the other chromosome. To better understand the complex regulation of exon 9 splicing, we analyzed the levels of correctly spliced CFTR transcripts in six CFTR-expressing epithelial cell lines derived from lung, colon, testis, vas deferens, and epididymis transiently transfected with four CFTR minigenes (pTG11T7, pTG12T7, pTG12T5, and pTG12T3). In this work, we show that a decrease in the Ts at the polymorphic locus in a TG12 background determines a cell-type dependent reduction in exon 9+ transcripts that is not related to the basal splicing efficiency in the cell line. These data emphasize the role of the T5 allele in CBAVD and identify the T3 allele as a severe cystic fibrosis (CF) disease-causing mutation. Finally, UV cross-linking experiments demonstrated that tissue-specific trans -acting splicing factors do not contribute to the different patterns of exon 9 splicing found between the cell lines. However, we observed that lower numbers of Ts can alter the binding of TDP-43 (TDP43 or TARDBP) to its specific target ug12 in a tissue-specific manner. Our results support the idea that the ratio of general splicing factors plays a role in the tissue variability of exon 9 alternative splicing. Hum Mutat 25:72,81, 2005. © 2004 Wiley-Liss, Inc. [source] Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2004Sofía Fraile Summary The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined. A strong dimerization domain (i.e. an amphipathic ,-helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope-tagged version of the 23 kDa C-terminal secretion signal of haemolysin (EHlyA). The zipper-containing polypeptide (ZEHlyA) was effectively secreted by E. coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography. In vivo protein cross-linking experiments and coexpression with a secretion-deficient derivative of ZEHlyA indicated that leucine zipper-dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin VHH -domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the VHH moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both VHH -EHlyA and VHH -ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric VHH -ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10-fold increase in their avidity. These data reveal a non-anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity. [source] Miranda cargo-binding domain forms an elongated coiled-coil homodimer in solution: Implications for asymmetric cell division in DrosophilaPROTEIN SCIENCE, Issue 5 2008Mohammad S. Yousef Abstract Miranda is a multidomain adaptor protein involved in neuroblast asymmetric division in Drosophila melanogaster. The central domain of Miranda is necessary for cargo binding of the neural transcription factor Prospero, the Prospero-mRNA carrier Staufen, and the tumor suppressor Brat. Here, we report the first solution structure of Miranda central "cargo-binding" domain (residues 460,660) using small-angle X-ray scattering. Ab initio modeling of the scattering data yields an elongated "rod-like" molecule with a maximum linear dimension (Dmax) of ,22 nm. Moreover, circular dichroism and cross-linking experiments indicate that the cargo-binding domain is predominantly helical and forms a parallel coiled-coil homodimer in solution. Based on the results, we modeled the full-length Miranda protein as a double-headed, double-tailed homodimer with a long central coiled-coil region. We discuss the cargo-binding capacity of the central domain and propose a structure-based mechanism for cargo release and timely degradation of Miranda in developing neuroblasts. [source] Probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structurePROTEIN SCIENCE, Issue 12 2004Xiaoduan Ye Abstract Emerging high-throughput techniques for the characterization of protein and protein-complex structures yield noisy data with sparse information content, placing a significant burden on computation to properly interpret the experimental data. One such technique uses cross-linking (chemical or by cysteine oxidation) to confirm or select among proposed structural models (e.g., from fold recognition, ab initio prediction, or docking) by testing the consistency between cross-linking data and model geometry. This paper develops a probabilistic framework for analyzing the information content in cross-linking experiments, accounting for anticipated experimental error. This framework supports a mechanism for planning experiments to optimize the information gained. We evaluate potential experiment plans using explicit trade-offs among key properties of practical importance: discriminability, coverage, balance, ambiguity, and cost. We devise a greedy algorithm that considers those properties and, from a large number of combinatorial possibilities, rapidly selects sets of experiments expected to discriminate pairs of models efficiently. In an application to residue-specific chemical cross-linking, we demonstrate the ability of our approach to plan experiments effectively involving combinations of cross-linkers and introduced mutations. We also describe an experiment plan for the bacteriophage , Tfa chaperone protein in which we plan dicysteine mutants for discriminating threading models by disulfide formation. Preliminary results from a subset of the planned experiments are consistent and demonstrate the practicality of planning. Our methods provide the experimenter with a valuable tool (available from the authors) for understanding and optimizing cross-linking experiments. [source] | |||||||||||||