The refolding and reassembly of Escherichia coli heat-labile enterotoxin B-subunit: analysis of reassembly-competent and reassembly-incompetent unfolded states

The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is an exceptionally stable protein maintaining its quaternary structure over the pH value range 2.0-11.0. Up to 80% yields of reassembled pentamer can be obtained in vitro from material disassembled for very short incubation periods in KCl-HCl, pH 1.0. However, when the incubation period in acid is extended, the reassembly yield decreases to no more than 20% (Ruddock et al. (1996) J. Biol. Chem. 271 19118-19123). Here we demonstrate that the ion species present in the disassembly conditions strongly influence the reassembly competence of EtxB showing that 60% reassembly yields can be achieved, even after prolonged incubations, by the use of a phosphate buffer for acid disassembly. Using this system, we have fully characterized the disassembly and reassembly behavior of EtxB by electrophoretic, immunochemical, and spectroscopic techniques and compared it with that previously observed. Depending on the denaturation system used, the acid-denatured monomer is either in a predominantly reassembly-competent state (H(3)PO(4) system) or in a predominantly reassembly-incompetent conformation (KCl-HCl system). Interconversion between these two conformations in the denatured state is possible by the addition of salts to the denatured protein. The results are consistent with the previous hypothesis that the conversion between reassembly-competent and -incompetent states corresponds to a cis/trans isomerization of a peptide bond, presumably that to Pro93.     

Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation

Genes belonging to the Nkx, Gsh and Msx families are expressed in similar dorsovental spatial domains of the insect and vertebrate central nervous system (CNS), suggesting the bilaterian ancestor used this genetic program during CNS development. We have investigated the significance of these similar expression patterns by testing whether Nkx6 proteins expressed in ventral CNS of zebrafish and flies have similar functions. In zebrafish, Nkx6.1 is expressed in early-born primary and later-born secondary motoneurons. In the absence of Nkx6.1, there are fewer secondary motoneurons and supernumerary ventral interneurons, suggesting Nkx6.1 promotes motoneuron and suppresses interneuron formation. Overexpression of fish or fly Nkx6 is sufficient to generate supernumerary motoneurons in both zebrafish and flies. These results suggest that one ancestral function of Nkx6 proteins was to promote motoneuron development.     

Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group

Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hemoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme.     

Expression,purification and characterisation of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI

The fer gene from Bacillus subtilis has been subcloned and overexpressed in Escherichia coli and the protein (Fer) purified to homogeneity. N-Terminal sequencing and mass spectrometry indicate that the initiator methionine is removed from the protein and that the molecular mass is 8732 Da consistent with that deduced from the gene sequence. Amino-acid sequence comparisons indicate that Fer is a ferredoxin containing a 4Fe-4S cluster. The electron paramagnetic resonance spectrum of the reduced form of Fer is typical for a [4Fe-4S](+) cluster showing rhombic signals with g values of 2.07, 1.93 and 1.88. Reduced Fer also gives rise to a magnetic circular dichroism spectrum typical of a [4Fe-4S](+) cluster. Potentiometric titrations indicate that Fer has a reduction potential of -385+/-10 mV for the [4Fe-4S](+)-[4Fe-4S](2+) redox couple, well within the normal range expected for such a ferredoxin. A proposed physiological role for Fer is as an electron donor to cytochrome P450 BioI. Studies on Fer binding to P450 BioI give rise to a K(d) value of 0.87+/-0.10 microM. Anaerobic experiments using CO-saturated buffer indicate that Fer is indeed capable of transferring electrons to this cytochrome P450 albeit at a fairly low rate.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Molecular and spectroscopic analysis of the cytochrome cbb(3) oxidase from Pseudomonas stutzeri

Cytochrome cbb(3) oxidase, a member of the heme-copper oxidase superfamily, is characterized by its high affinity for oxygen while retaining the ability to pump protons. These attributes are central to its proposed role in the microaerobic metabolism of proteobacteria. We have completed the first detailed spectroscopic characterization of a cytochrome cbb(3) oxidase, the enzyme purified from Pseudomonas stutzeri. A combination of UV-visible and magnetic CD spectroscopies clearly identified four low-spin hemes and the high-spin heme of the active site. This heme complement is in good agreement with our analysis of the primary sequence of the ccoNOPQ operon and biochemical analysis of the complex. Near-IR magnetic CD spectroscopy revealed the unexpected presence of a low-spin bishistidine-coordinated c-type heme in the complex. This was shown to be one of two c-type hemes in the CcoP subunit by separately expressing the subunit in Escherichia coli. Separate expression of CcoP also allowed us to unambiguously assign each of the signals associated with low-spin ferric hemes present in the X-band EPR spectrum of the oxidized enzyme. This work both underpins future mechanistic studies on this distinctive class of bacterial oxidases and raises questions concerning the role of CcoP in electron delivery to the catalytic subunit.     

SEGMENTATION APPROACHES THAT DIFFERENTIATE CONSUMPTION FREQUENCY FROM SENSORY PREFERENCE1

People can eat a food without having a strong preference for it, and people can prefer a food without eating it. Given this seeming disconnect between attitude and behavior, which type of measure or segment can best be used to profile or identify loyal consumer segments of a food, such as soy? This research compares a usage‐based method (heavy‐light‐nonusers) with a new attitude‐based method (seeker‐neutral‐avoider), and finds that the attitude‐based method differentiates purchase‐related intentions better than the usage‐based method. Implications for profiling consumer taste patterns and consumer segments are provided.     

Selective advantages created by codon ambiguity allowed for the evolution of an alternative genetic code in Candida spp

Several species of the genus Candida decode the standard leucine CUG codon as serine. This and other deviations from the standard genetic code in both nuclear and mitochondrial genomes invalidate the notion that the genetic code is frozen and universal and prompt the questions ‘why alternative genetic codes evolved and, more importantly, how can an organism survive a genetic code change?’ To address these two questions, we have attempted to reconstruct the early stages of Candida albicans CUG reassignment in the closely related yeast Saccharomyces cerevisiae. These studies suggest that this genetic code change was driven by selection using a molecular mechanism that requires CUG ambiguity. Such codon ambiguity induced a significant decrease in fitness, indicating that CUG reassignment can only be selected if it introduces an evolutionary edge to counteract the negative impact of ambiguity. We have shown that CUG ambiguity induces the expression of a novel set of stress proteins and triggers the general stress response, which, in turn, creates a competitive edge under stress conditions. In addition, CUG ambiguity in S. cerevisiae induces the expression of a number of novel phenotypes that mimic the natural resistance to stress characteristic of C. albicans. The identification of an evolutionary advantage created by CUG ambiguity is the first experimental evidence for a genetic code change driven by selection and suggests a novel role for codon reassignment in the adaptation to new ecological niches.     

Characterization and molecular genetic complementation of mutants affecting dimorphism in the fungus ustilago maydis

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase, uac1. Secondary mutagenesis of a uac1 disruption strain allowed the isolation of a large number of suppressor mutants, termed ubc, for Ustilago bypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of the ubc1 gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three new ubc genes, termed ubc2, ubc3, and ubc4. We also describe the morphology of the ubc2, ubc3, and ubc4 mutants in a uac1- background as well as in a background with a functional uac1 gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additional ubc genes. Copyright 1998 Academic Press.