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The effect of high concentration, also referred to as crowding conditions, on Brownian motion is of central relevance for the understanding of the physical, chemical and biological properties of proteins in their native environment. Specifically, the simple inverse relationship between the translational diffusion coefficient and the macroscopic solution viscosity as predicted by the generalized Stokes-Einstein (GSE) relation has been the subject of many studies, yet a consensus on its applicability has not been reached. Here, we use isotope-filtered pulsed-field gradient NMR to separately assess the μm-scale diffusivity of two proteins, BSA and an SH3 domain, in mixtures as well as single-protein solutions, and demonstrate that transient binding can account for an apparent violation of the GSE relation. Whereas GSE behavior applies for the single-protein solutions, it does not hold for the protein mixtures. Transient binding behavior in the concentrated mixtures is evidenced by calorimetric experiments and by a significantly increased apparent activation energy of diffusion. In contrast, the temperature dependence of the viscosity, as well as of the diffusivity in single-component solutions, is always dominated by the flow activation energy of pure water. As a practically relevant second result, we further show that, for high protein concentrations, the diffusion of small molecules such as dioxane or water is not generally a suitable probe for the viscosity experienced by the diffusing proteins.

ABSTRACTTo study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated withEscherichia coliK-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated inE. coliisolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression ofahpCFanddps, which was not observed in anE. coliΔoxyRmutant. Growth ofahpCFandoxyRdeletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation ofE. colito osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinalE. colito better cope with diet-induced osmotic stress.

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Poly-E-caprolactam (Nylon-6) wurde direkt in der Ionenquelle eines Massenspektrometers pyrolysiert. Bereits bei Temperaturen unterhalb 100°C beginnen dabei die im Polymeren enthaltenen cyclischen Oligomeren im Hochvakuum zu verdampfen und konnen massenspektrometrisch ohne vorherige Isolierung identifEiert werden. Bei Temperaturen oberhalb 390°C wird unter den genannten Bedingungen das Einsetzen eines intensiven thermischen Abbaus beobachtet, und es kann gezeigt werden, daD hierbei iiberwiegend cyclische Oligomere des Caprolactams gebildet werden. SUMMARY:Poly-E-caprolactam had been pyrolyzed directly in the ion source of a mass spectrometer. Already at temperatures below 100°C cyclic oligomers present in the polymer begin to evaporate in the high vacuum and can be identified mass spectrometrically without previous isolation. At temperatures above 390°C the start of an intensive thermal degradation is observed under the described conditions, and it can be shown that mainly cyclic oligomers of caprolactam are produced.

Ulm, Federal Republic of Gennany SynopsisCombinations of L-and D-proline residues are useful compounds for finding new structures and properties of cyclic peptides. This is demonstrated with one striking example, the cyclic tetrapep-For this molecule composed of strictly alternating D-and L-configurated residues, a highly symmetrical structure is expected, which should be an optically inactive meso-form. Cyclization of the enantiomeric pure linear precursor D-P~o-L-P~o-D-P~o-L-P~o, however, yields a racemic mixture of two enantiomeric cyclotetrapeptides, both with twofold symmetry and a cis-trans-cis-trans sequence of the peptide bonds. Remarkably, this formation of a racemate was not caused by racemization, but by &/trans isomerization of all peptide bonds in the ring. This procans may occur in the,linear precursor during the ring formation (cyclization of conformers with trans-cis-trans or cis-trans-cis arrangement of the amide bonds) as well as in the enantiomeric pure cyclic tetrapeptide at higher temperature. In the latter case, an all& structure should exist as the intermediate, which can form a cis-truns-cis-truns sequence in two equivalent ways, leading finally to two enantiomeric cyclotetrapeptides. In the first one, the cis peptide bonds are attributed to the L-residues and the trans peptide bonds to the &residues; in the second one, the cis bonds belong to the D and the trans bonds to the bresidues. The mixture of these two enantiomers does not crystallize in the racemic form, but in enantiomeric pure separate crystals. The structural properties could be proved by 'H-and 13C-nmr spectroscopy and x-ray analysis. The cis/truns isomerization process was confirmed by optical rotation measurements and CD spectroscopy, as well as DREIDING model studies. Calorimetric measurements in the solid state suggest the existence of the expected all-cis intermediate. The backbone conformation of the 12-membered medium-sized ring shows only slight deviations-up to 6"-from the planarity of the peptide bonds. On the other hand, the four pyrrolidine rings show different types of puckering of the C, or the C, atoms.

Sulfur compounds are generally accepted as being of high importance for sensory perception of meat flavour. The main problems of sulfur compounds' analysis are low flavour thresholds indicating high aroma effectiveness, high lability providing transformation into secondary products as well as active interaction with various organic substances present in meat systems. Probable pathways for formation and degradation of sulfur compounds are demonstrated.Sulfur compounds were isolated from volatiles of boiled beef by precipitation with mercuric chloride and regeneration. The separation of volatile amines and carbonyl compounds by derivatization remarkably improved the aroma of the samples.Gas chromatographic identification with flame ionization and flame photometric detectors was accomplished on the base of retention index comparison on 4 glass columns of different polarity. Most of the peaks detected by FID were found to have identical index values in three different samples of beef. The comparison of the identification results with those of volatiles isolated from MAILLARD reaction products providing heated meat flavour, show large differences. It could be stated that regeneration products from mercuric chloride precipitates besides of sulfur compounds contain oxygen components as well as saturated and unsaturated hydrocarbons, too.