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Domain Mutants (domain + mutant)
Selected AbstractsLipophilic regulator of a developmental switch in Caenorhabditis elegansAGING CELL, Issue 6 2004Matthew S. Gill In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulin-like and transforming growth factor , (TGF,)-like signalling pathways. It has been proposed that lipophilic hormones act downstream of these pathways to regulate dauer formation. One likely target for such a hormone is DAF-12, an orphan nuclear hormone receptor that mediates these developmental decisions and also influences adult lifespan. In order to find lipophilic hormones we have generated lipophilic extracts from mass cultures of C. elegans and shown that they rescue the dauer constitutive phenotype of class 1 daf-2 insulin signalling mutants and the TGF, signalling mutant daf-7. These extracts are also able to rescue the lethal dauer phenotype of daf-9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF-12 ligand; extracts, however, have no effect on a DAF-12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF-9 dependent as extracts from a daf-9;daf-12 double mutant do not exhibit this activity. Preliminary fractionation of the lipophilic extracts shows that the activity is hydrophobic with some polar properties, consistent with a small lipophilic hormone. We propose that the dauer rescuing activity is a hormone synthesized by DAF-9 that acts through DAF-12. [source] Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasmaFEBS JOURNAL, Issue 22 2009Michael J. Krisinger Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P10S12D23Q32N33) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca2+ titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca2+ requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity. [source] Functional analysis of the heavy metal binding domains of the Zn/Cd-transporting ATPase, HMA2, in Arabidopsis thalianaNEW PHYTOLOGIST, Issue 1 2009Chong Kum Edwin Wong Summary ,,The Zn/Cd-transporting ATPase, HMA2, has N- and C-terminal domains that can bind Zn ions with high affinity. Mutant derivatives were generated to determine the significance of these domains to HMA2 function in planta. ,,Mutant derivatives, with and without a C-terminal GFP tag, were expressed from the HMA2 promoter in transgenic hma2,hma4, Zn-deficient, plants to test for functionality. ,,A deletion mutant lacking the C-terminal 244 amino acids rescued most of the hma2,hma4 Zn-deficiency phenotypes with the exception of embryo or seed development. Root-to-shoot Cd translocation was fully rescued. The GFP-tagged derivative was partially mis-localized in the root pericycle cells in which it was expressed. Deletion derivatives lacking the C-terminal 121 and 21 amino acids rescued all phenotypes and localized normally. N-terminal domain mutants localized normally but failed to complement the hma2,hma4 phenotypes. ,,These observations suggest that the N-terminal domain of HMA2 is essential for function in planta while the C-terminal domain, although not essential for function, may contain a signal important for the subcellular localization of the protein. [source] Hydrophile scanning as a complement to alanine scanning for exploring and manipulating protein,protein recognition: Application to the Bim BH3 domainPROTEIN SCIENCE, Issue 7 2008Melissa D. Boersma Abstract Alanine scanning has been widely employed as a method of identifying side chains that play important roles in protein,protein and protein,peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild-type residue to alanine removes most of the side-chain atoms, and the effect of this removal is typically interpreted to indicate contribution of the deleted side chain to the stability of the complex. Hydrophile scanning involves systematic mutation of wild-type residues to a cationic or anionic residue (lysine or glutamic acid, in this case). We find that the results of these mutations provide insights on interactions between polypeptide surfaces that are complementary to the information obtained via alanine scanning. We have applied this technique to a peptide that corresponds to the BH3 domain of the pro-apoptotic protein Bim. The wild-type Bim BH3 domain binds strongly to the anti-apoptotic proteins Bcl-xL and Mcl-1. Combining information from the alanine, lysine, and glutamic acid scans has enabled us to identify Bim BH3 domain mutants containing only two or three sequence changes that bind very selectively either to Bcl-xL or Mcl-1. Our findings suggest that hydrophile scanning may prove to be a broadly useful tool for revealing sources of protein,protein recognition and for engineering selectivity into natural sequences [source] Domain,Domain Interactions in the Iterative Type I Polyketide Synthase ATX from Aspergillus terreusCHEMBIOCHEM, Issue 8 2008Tomomi Moriguchi Analyzing ATX mutants: ATX, a 6-methylsalicylic acid synthase from Aspergillus terreus, has five catalytic domains in its monomer and forms a homotetramer. Coexpression of inactive ATX catalytic domain mutants in yeast showed that ATX activity was reconstituted by all combinations of the domain mutants, suggesting that the five catalytic domains could interact with each other with substantial flexibility to carry out 6-methylsalicylic acid synthesis. [source] | |||||||||||||