Home About us Contact
|
Home About us Contact | ||
|
|
||
Construct Consisting (construct + consisting)
Selected AbstractsMeasurement of dissociation rate of biomolecular complexes using CEELECTROPHORESIS, Issue 3 2009Peilin Yang Abstract Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide,protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of,53,s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30,cm long. When using Fyn-SH2-SH3 tagged with glutathione S -transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7,s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0±2.7 and 58.8±6.1,s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that although all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions. [source] Nas transgenic mouse line allows visualization of Notch pathway activity in vivoGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2006Céline Souilhol Abstract The Notch signaling pathway plays multiple and important roles in mammals. However, several aspects of its action, in particular, the precise mapping of its sites of activity, remain unclear. To address this issue, we generated a transgenic line carrying a construct consisting of a nls-lacZ reporter gene under the control of a minimal promoter and multiple RBP-J, binding sites. Here we show that this transgenic line, which we termed NAS (for Notch Activity Sensor), displays an expression profile that is consistent with current knowledge on Notch activity sites in mice, even though it may not report on all these sites. Moreover, we observe that NAS transgene expression is abolished in a RBP-J,-deficient background, indicating that it indeed requires Notch/RBP-J, signaling pathway activity. Thus, the NAS transgenic line constitutes a valuable and versatile tool to gain further insights into the complex and various functions of the Notch signaling pathway. genesis 44: 277,286, 2006. © 2006 Wiley-Liss, Inc. [source] Structure of the N-terminal fragment of Escherichia coli Lon proteaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Mi Li The structure of a recombinant construct consisting of residues 1,245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6,Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal ,-helix. The structure of the first subdomain (residues 1,117), which consists mostly of ,-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates. [source] Cytoplasmic tail motifs mediate endoplasmic reticulum localization and export of transmembrane reporters in the protozoan parasite Toxoplasma gondiiCELLULAR MICROBIOLOGY, Issue 6 2000Heinrich C. Hoppe In mammalian cells and yeasts, amino acid motifs in the cytoplasmic tails of transmembrane proteins play a prominent role in protein targeting in the early secretory pathway by mediating localization to or rapid export from the endoplasmic reticulum (ER). However, early sorting events are poorly characterized in protozoan parasites. Here, we show that a C-terminal QKTT sequence mediates the ER localization of chimeric reporter constructs consisting of bacterial alkaline phosphatase (BAP) fused to the transmembrane domain (TMD) and truncated cytoplasmic tail of the human low-density lipoprotein receptor (LDL) receptor or of murine lysosome-associated membrane protein (lamp-1) in Toxoplasma gondii. The cytoplasmic tail of human TGN46 also determines ER localization of BAP chimeras in the parasite, but this can be overcome by the addition at the C-terminus of the tail of an acidic patch, which functions as an ER export signal in conjunction with an upstream tyrosine motif. These results suggest that COPI-dependent ER retrieval and COPII-dependent export mechanisms mediated by KKXX and DXE motifs of mammalian cells are generally conserved in T. gondii. In contrast, the failure of the QKTT motif and TGN46 cytoplasmic tail to induce steady-state ER localization of vesicular stomatitis virus glycoprotein (VSVG) chimeras in HeLa and NRK cells indicates that significant differences in early secretory trafficking also exist. [source] | ||||||||||