Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry     

Structural and physicochemical requirements of endotoxins for the activation of arachidonic acid metabolism in mouse peritoneal macrophages in vitro

Lipopolysaccharides of different wild-type and mutant gram-negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep-rough mutants of Salmonella minnesota and Escherichia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC4) and prostaglandin E2 (PGE2). Lipopolysaccharides of wild-type strains (S. abortus equi, S. friedenau) only induced PGE2 and not LTC4 formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC4 and PGE2 production. Deacylated Re-mutant lipopolysaccharide preparations were inactive. However, co-incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC4 and PGE2, similar to those obtained with Re-mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin-low-responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re-mutant lipopolysaccharide and that polymyxin B prevented the Re-lipopolysaccharide-induced LTC4 and PGE2 release. Physical measurements showed that the phase-transition temperatures of both free lipid A and S-form lipopolysaccharide were above 37 degrees C while those of R-mutant lipopolysaccharides were significantly lower (30-35 degrees C). Thus, with the materials investigated, an inverse relationship between the phase-transition temperature and the capacity to elicit LTC4 production was revealed.     

Phosphorylation of an 85-kd membrane protein by a novel mechanism

Protein phosphorylation has been recognized as a major mechanism for the regulation of cellular functions. The classical phosphate donor in protein phosphorylation reactions is ATP. Here we show that 3'-phosphoadenosine-5'-phosphosulphate (PAPS), a ubiquitous nucleotide so far known to have a central role in sulphate transfer, serves as phosphate donor for protein phosphorylation. In a very specific, rapid and probably autocatalytic reaction, the 3'-phosphate group of PAPS was found to be transferred to a serine residue of an 85-kd membrane protein (p85). ATP did not serve as phosphate donor in this reaction. Radioactive phosphate incorporated into p85 in a membrane fraction was rapidly lost by dephosphorylation after removal of PAPS or by exchange with unlabelled phosphate after addition of nonradioactive PAPS. PAPS-dependent phosphorylation of the 85-kd protein and other proteins was observed in all rat and bovine tissues examined, as well as in various mammalian cell lines. Our results indicate the existence of a novel widespread form of protein phosphorylation.     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

Planar asymmetric lipid bilayers of glycosphingolipid or lipopolysaccharide on one side and phospholipids on the other: membrane potential, porin function, and complement activation.

We have determined some physicochemical properties of the monosaccharide-type fraction (GSL-1) of glycosphingolipids, the major glycolipid components of the outer leaflet of the Gram-negative species Sphingomonas paucimobilis. These properties included the state of order of the hydrocarbon moiety, the effective molecular area, surface charge density, and intrinsic transmembrane potential profile of reconstituted planar asymmetric GSL-1/phospholipid bilayer membranes. We have, furthermore, investigated the insertion into and the function of porin channels in the reconstituted bilayers and the complement-activating capability of GSL-1 surfaces. All results were compared with respective data for deep rough mutant lipopolysaccharide of Salmonella minnesota R595. We found a remarkable agreement in most functional properties of the two glycolipids.     

A 28,000-Da GDP/GTP-binding protein specific to the nuclear envelope

Using a photoaffinity labeling approach to characterize GDP/GTP-binding proteins of the nuclear envelope, we found that several polypeptides of isolated rat liver nuclear envelopes were photolabeled with [alpha-32P]GDP. One of these labeled proteins, which had an apparent molecular mass of 28 kDa (p28), was highly enriched in nuclear envelopes relative to unfractionated nuclei and was not detectable in fractions of mitochondria, cytosol, and endoplasmic reticulum membranes by two-dimensional gel electrophoresis. Thus p28 is specific to the nuclear envelope. Chemical extractions showed that p28 was tightly associated with a subfraction of the nuclear envelope enriched in nuclear pore complexes and lamina. Solubilization of p28 required buffers containing 1% octylglucoside and at least 300 mM KCl. Photolabeling of p28 was specific for GTP and GDP, since no other guanine or adenine nucleotide tested was able to compete efficiently with [alpha-32P]GDP for photolabeling. Added magnesium or manganese cations were not required for photolabeling of p28, and labeling was substantially inhibited with 10 mM MgCl2, 1 mM MnCl2, and KCl concentrations greater than 150 mM. Sucrose density centrifugation showed that detergent-solubilized p28 had a S value of 2.5. The properties of p28 described in this study suggest that it may have a role in functions of the pore complex or lamina.     

Positive Facial Affect – An fMRI Study on the Involvement of Insula and Amygdala

Imitation of facial expressions engages the putative human mirror neuron system as well as the insula and the amygdala as part of the limbic system. The specific function of the latter two regions during emotional actions is still under debate. The current study investigated brain responses during imitation of positive in comparison to non-emotional facial expressions. Differences in brain activation of the amygdala and insula were additionally examined during observation and execution of facial expressions. Participants imitated, executed and observed happy and non-emotional facial expressions, as well as neutral faces. During imitation, higher right hemispheric activation emerged in the happy compared to the non-emotional condition in the right anterior insula and the right amygdala, in addition to the pre-supplementary motor area, middle temporal gyrus and the inferior frontal gyrus. Region-of-interest analyses revealed that the right insula was more strongly recruited by (i) imitation and execution than by observation of facial expressions, that (ii) the insula was significantly stronger activated by happy than by non-emotional facial expressions during observation and imitation and that (iii) the activation differences in the right amygdala between happy and non-emotional facial expressions were increased during imitation and execution, in comparison to sole observation. We suggest that the insula and the amygdala contribute specifically to the happy emotional connotation of the facial expressions depending on the task. The pattern of the insula activity might reflect increased bodily awareness during active execution compared to passive observation and during visual processing of the happy compared to non-emotional facial expressions. The activation specific for the happy facial expression of the amygdala during motor tasks, but not in the observation condition, might reflect increased autonomic activity or feedback from facial muscles to the amygdala.     

Beautiful,complicated—and intelligent? Novel aspects of the thigmonastic stamen movement in Loasaceae

In a recent study we investigated the complex mechanisms regulating the pollen release via thigmonastic stamen movement found exclusively in Loasaceae subfamily Loasoideae. We demonstrated that stamen movement is modulated by abiotic (light and temperature) as well as biotic stimuli (pollinator availability and visitation frequency). This is explained as a mechanism to adjust the rate of stamen movement and thus pollen dispensation to different environmental conditions in order to optimize pollen transfer. Stamen movement is rapid and thus a near-immediate response to pollinator visits. However, Loasaceae flowers also show a response to biotic stimuli on a longer time scale, by adjusting the duration of both the staminate and the carpellate phase of the anthesis. We here present two additional data sets on species not previously studied, underscoring the shortening of the staminate phase in the presence of pollinator visits vs. their absence and the shortening of the carpellate phase after pollination. Overall, the plant shows not only a rapid but an “intelligent” reaction to its environment in adjusting anthesis and pollen presentation to a range of factors. The physiological and morphological bases of the stamen movement are poorly understood. Our previous study showed that there is no direct spatial relationship between the place of stimulation in the flower and the stamen bundle activated. We here further show the morphological basis for stamen movement from a reflexed into an erect position: Only the basal part of the filament curves around the receptacle, while the upper part of the filament retains its shape. We hypothesize that the stimulus is transmitted over the entire receptacle and the place of reaction is determined by stamen maturity, not the location of the stimulus.