Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind cooperatively to actin and regulate myosin.

Tropomyosin (Tm) is an evolutionarily conserved ?-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilising actin-filaments and regulate myosin function. The fission yeast, Schizosaccharomyces pombe, possesses a single essential Tm, Cdc8, which can be acetylated on its amino terminal methionine to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with amino terminal substitutions to explore how stability of the Cdc8-polymer overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown the stability of the amino terminal of the Cdc8 ?-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased amino-terminal stability, dimers of which are capable of forming Tm-polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild type, and was unable to regulate actomyosin interactions. The data presented here are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin-filament, consistent with a cooperative model for Tm binding to actin

Live Cell Imaging in Fission Yeast

Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast

Formins Determine the Functional Properties of Actin Filaments in Yeast

The actin cytoskeleton executes a broad range of essential functions within a living cell. The dynamic nature of the actin polymer is modulated to facilitate specific cellular processes at discrete locations by actin-binding proteins (ABPs), including the formins and tropomyosins (Tms). Formins nucleate actin polymers, while Tms are conserved dimeric proteins that form polymers along the length of actin filaments. Cells possess different Tm isoforms, each capable of differentially regulating the dynamic and func- tional properties of the actin polymer. However, the mecha- nism by which a particular Tm localizes to a specific actin polymer is unknown. Here we show that specific formin family members dictate which Tm isoform will associate with a particular actin filament to modulate its dynamic and functional properties at specific cellular locations. Exchanging the localization of the fission yeast formins For3 and Cdc12 results in an exchange in localizations of Tm forms on actin polymers. This nucleator-driven switch in filament composition is reflected in a switch in actin dynamics, together with a corresponding change in the filament’s ability to regulate ABPs and myosin motor activity. These data establish a role for formins in dictating which specific Tm variant will associate with a growing actin filament and therefore specify the functional capacity of the actin filaments that they create

Influence of emulsifier type on the spray-drying properties of model infant formula emulsions

The objective of this study was to compare the drying performance and physicochemical properties of model infant formula (IF) emulsions containing 43, 96 and 192 g L−1 protein, oil and maltodextrin (MD), respectively, prepared using different emulsifier systems. Emulsions were stabilised using either whey protein isolate (WPI), whey protein hydrolysate (WPH; DH 8%), WPH + CITREM (9 g L−1), WPH + lecithin (5 g L−1) or WPH conjugated with maltodextrin (DE 12) (WPH-MD). Homogenised emulsions had 32% solids content and oil globules with mean volume diameter WPH + LEC > WPH > WPH- MD > WPI, WPI > WPH > WPH- MD > WPH + LEC > WPH + CIT and WPH- MD > WPI > WPH > WPH + LEC > WPH + CIT, respectively. Additionally, differences in wettability, surface topography and oil globule distribution within the powder matrix and in reconstituted powders were linked to the emulsifier system used. Inclusion of the WPH-MD conjugate in the formulation of IF powder significantly improved drying behaviour and physicochemical properties of the resultant powder, as evidenced by lowest powder build-up during drying and greatest emulsion quality on reconstitution, compared to the other model formula systems

Antibacterial compounds

The present invention relates to certain compounds of formula (I) wherein - Ar is (Ar1) or (Ar2) and to their uses as antibacterial agents. The invention further relates to methods of treatment of bacterial infection with such compounds, optionally in combination with other antimicrobials and to compositions and pharmaceutical formulations containing such compounds. The invention additionally relates to coatings containing such compounds and to items having such coatings

A review of the analytical approaches used for studying the structure, interactions and stability of emulsions in nutritional beverage systems

Nutritional beverage emulsions contain water and oil, stabilised by surfactants, and are both diverse and complex. Their susceptibility to changes induced by manufacturing processes and on storage, results in challenges with their stability, quality and shelf-life. An understanding of the relationship between structure and stability of an emulsion is essential to designing and competently formulating food products with the desired nutritional functionality and sensory properties, while achieving the required shelf-life. This article critically reviews a selection of commonly-used analytical approaches focused on characterisation of emulsion structure in the context of emulsion formation, techno-functional properties and stability to intrinsic and environmental factors

Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol

Optimised production and analysis of recombinant protein-filled vesicles from E. coli.

This protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulphide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to many areas of scientific research including applied biotechnology and medicine. This innovative system, using a short peptide tag, that exports diverse recombinant proteins in membrane bound vesicles from E. coli 1 , provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulphide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture media and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine. In this article, and associated video, a detailed protocol of the method is provided, and highlights key steps in the methodology to maximise recombinant protein filled vesicle production

Identification of sequence changes in myosin II that adjust muscle contraction velocity.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (β/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of β-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human β-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human β-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of β-myosin as species mass increased to enable a reduced contraction velocity and heart rate