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Substitution Mutants (substitution + mutant)

Distribution by Scientific Domains

Chemistry	50%

Selected Abstracts

Assembly of the Rieske iron,sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplasts

FEBS JOURNAL, Issue 2 2000
Aliki Kapazoglou
The assembly of the Rieske iron,sulphur protein into the cytochrome bf complex was examined following import of 35S-labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe-2S] centre; the mutant ,45,52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lumenal domain; and mutants ,168,173 and ,177,179 which removed two C-terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe,2S] cluster, the glycine-rich region and the C-terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts. [source]

Analysis of the requirements for pilus biogenesis at the outer membrane usher and the function of the usher C-terminus

MOLECULAR MICROBIOLOGY, Issue 2 2006
Stephane Shu Kin So
Summary Uropathogenic strains of Escherichia coli assemble type 1 and P pili to colonize the bladder and kidney respectively. These pili are prototype structures assembled by the chaperone/usher secretion pathway. In this pathway, a periplasmic chaperone works together with an outer membrane (OM) usher to control the folding of pilus subunits, their assembly into a pilus fibre and secretion of the fibre to the cell surface. The usher serves as the assembly and secretion platform in the OM. The usher has distinct functional domains, with the N-terminus providing the initial targeting site for chaperone,subunit complexes and the C-terminus required for subsequent stages of pilus biogenesis. In this study, we investigated the molecular interactions occurring at the usher during pilus biogenesis and the function of the usher C-terminus. We provide genetic and biochemical evidence that the usher functions as a complex in the OM and that interaction of the pilus adhesin with the usher is critical to prime the usher for pilus biogenesis. Analysis of C-terminal truncation and substitution mutants of the P pilus usher PapC demonstrated that the C-terminus is required for proper binding of chaperone,subunit complexes to the usher and plays an important role in assembly of complete pili. [source]

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a ,-barrel structure

MOLECULAR MICROBIOLOGY, Issue 1 2005
Angela Valeva
Summary Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a ,-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel ,-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the ,-barrel-forming sequence of staphylococcal cytolysins and identify the ,-barrel as a membrane-perforating structure that is highly conserved in evolution. [source]

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity

PROTEIN SCIENCE, Issue 12 2001
Anne Chapman-Smith
BCCP, biotin carboxyl carrier protein; IPTG, isopropyl-1-thio-,-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; S.D., standard deviation Abstract Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5,-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain. [source]