Detection of protein–protein interactions by a combination of a novel cytoplasmic membrane targeting system of recombinant proteins and fluorescence resonance energy transfer

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)–protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein–protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP–protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP–protein A and EYFP–Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein–protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei. S. Shibasaki and K. Kuroda equally contributed to this work     

Increase in cell viability by polyamines through stimulation of the synthesis of ppGpp regulatory protein and ω protein of RNA polymerase in Escherichia coli

It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called "polyamine modulon". We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine-Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp-RpoZ complex.     

The lipid–teichoic acid complex in the cytoplasmic membrane of Streptococcus faecalis N.C.I.B. 8191

1. A lipid-teichoic acid complex was isolated from Streptococcus faecalis N.C.I.B. 8191. The covalent nature of the linkage between teichoic acid and lipid was established. 2. The complex exhibits macromolecular properties in solution, and ultracentrifugation studies show that these are due to micelle formation. 3. From chemical studies it is concluded that the teichoic acid is a poly(glycerol phosphate) in which some of the glycerol hydroxyl groups possess kojibiosyl [2-O-alpha-d-glucopyranosyl-(1-->2)-alpha-d- glucopyranosyl] substituents, together with d-alanine ester residues. 4. The lipid is 1-kojibiosyl diglyceride, already known as a membrane component of this organism, with probably a phosphatidyl substituent. The phosphatidyl kojibiosyl diglyceride is attached to the teichoic acid through a phosphodiester linkage, and the chain of the teichoic acid contains 28-35 units. 5. Although the complex represents the whole of the membrane teichoic acid in this organism, only about 12% of the membrane glycolipid is associated with teichoic acid. 6. Two phosphatidyl glycolipids, closely resembling that bearing the teichoic acid, were isolated from the lipids of the organism and were partly characterized.     

Unimolecular study of the interaction between the outer membrane protein OmpF from E. coli and an analogue of the HP(2–20) antimicrobial peptide

Little is known on antimicrobial peptide permeation through outer membrane channels in Gram-negative bacteria. Herein, we probed at a single-molecule level the interaction of two different peptides, magainin 2 and HPA3P with OmpF from E. coli. HPA3P is an analogue of the antimicrobial peptide HP(2–20) isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Our data show that the shorter and more charged HPA3P peptide is more accessible to the inner volume of the OmpF than magainin 2. We demonstrate the ability of HPA3P peptides to interact with OmpF in a voltage- and concentration-dependent manner, which does not rule out a novel mechanism by which such peptides could reach the periplasmic space of Gram-negative bacteria. Unexpectedly, we found that increasing the applied voltage led to an increase of the residence time of HPA3P peptide inside the pore, possibly reflecting electric field-induced changes in pore and peptide geometry.     

Progesterone 5β-reductase of Erysimum crepidifolium: cDNA cloning,expression in Escherichia coli,and reduction of enones with the recombinant protein

Erysimum is a genus of the Brassicaceae family closely related to the genus Arabidopsis. Several Erysimum species accumulate 5β-cardenolides. Progesterone 5β-reductases (P5βRs) first described in Digitalis species are thought to be involved in 5β-cardenolide biosynthesis. P5βRs belong to the dehydrogenase/reductase super-family of proteins. A full length cDNA clone encoding a P5βR was isolated from Erysimum crepidifolium leaves by 5′/3′ RACE-PCR (termed EcP5βR). Subsequently, the P5βR cDNAs of another nine Erysimum species were amplified by RT-PCR using 5′ and 3′ end primers deduced from the EcP5βR cDNA. The EcP5βR cDNA is 1170 bp long and encodes for 389 amino acids. The EcP5βR cDNA was ligated into the vector pQE 30 UA and the recombinant His-tagged protein (termed rEcP5βR) was over-expressed in Escherichia coli and purified by Ni-chelate affinity chromatography. Kinetic constants were determined for progesterone, 2-cyclohexen-1-one, isophorone, and NADPH. The by far highest specificity constant (k_cat K_M^?1) was estimated for 2-cyclohexen-1-one indicating that this monocyclic enone may be more related to the natural substrate of the enzyme than progesterone. The atomic structure of rEcP5βR was modelled using the crystal structure of P5βR from Digitalis lanata 2V6G as the template. All sequence motifs specific for SDRs as well as the NFYYxxED motif typical for P5βR-like enzymes were present and the protein sequence fitted into the template smoothly.     

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli��Expression and characterization of cytochrome-tagged Complex I subunits

Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane‐spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C‐terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c₅₅₀. Compared with other available fusion‐protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter‐like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo‐cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c₅₅₀ domain in all the fusion proteins exhibited normal spectra and redox properties, with an E_m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c‐tag. Finally, a his‐tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.     

Science,philosophy, and politics in the work of J. B. S. Haldane, 1922–1937

This paper analyzes the interaction between science, philosophy and politics (including ideology) in the early work of J. B. S. Haldane (from 1922 to 1937). This period is particularly important, not only because it is the period of Haldane's most significant biological work (both in biochemistry and genetics), but also because it is during this period that his philosophical and political views underwent their most significant transformation. His philosophical stance first changed from a radical organicism to a position far more compatible with mechanical materialism. The primary intellectual influence that was responsible for this shift was that of F. G. Hopkins. Later, Haldane came to accept Marxism and its official metaphysics, dialectical materialism, a move that let him accept the materialist conception of the world while still maintaining a resolute distance from mechanism. Throughout all these changes, what is most obvious is the influence of science on Haldane's philosophical views. An influence in the opposite direction is far less apparent.Parts of this paper are extracted from a longer work which concerns the interactions between philosophy and science throughout Haldane's scientific career (Sarkar forthcoming). The general conclusions reached here, from a consideration of Haldane's work only from 1922 to 1937 (see Section 6), remain the same for the rest of his life, as is detailed in the longer work. Thanks are due to R. S. Cohen, J. F. Crow, A. R. Fersht, J. Maynard Smith, R. C. Olby, D. Paul, M. Ruse, J. Stachel and S. Sturdy for helpful discussions and comments and criticism of the positions outlined in this paper. This is Contribution No. BTBG-92-4 from the Theoretical Biology Group, Boston University.