On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase.

Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.

Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study.

Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 A and 19 A, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.

Evidence for various calcium sites in human hair shaft revealed by sub-micrometer X-ray fluorescence.

New information about calcium status in human scalp hair shaft, deduced from X-ray micro-fluorescence imaging, including its distribution over the hair section, the existence of one or several binding-types and its variation between people, is presented. The existence of two different calcium types is inferred. The first one corresponds to atoms (or ions) easily removable by hydrochloric acid, located in the cortex (granules), in the cuticle zone and also in the core of the medulla, which can reasonably be identified as calcium soaps. The second type consists of non-easily removable calcium atoms (or ions) that are located in the medulla wall, probably also in the cuticle, and rather uniformly in the cortex; these calcium atoms may be involved in Ca(2+)-binding proteins, and their concentration is fairly constant from one subject to another. In addition to its nonuniform distribution across the hair section, the second striking feature of the first type calcium content is its high variability from one subject to another, by up to a factor 10. We expect this information to be useful for analyzing in more detail the relationship between hair calcium and environmental and medical factors.

Investigation of human hair cuticle structure by microdiffraction: direct observation of cell membrane complex swelling.

The cuticle of mammalian hair fibres protects the core of the fibre against physical and chemical stress. The structure and some of the properties of the cuticle have been extensively studied by electron microscopy. However, there is still a need for a less invasive structural probe. For this purpose, microdiffraction experiments have been carried out on human hair samples showing a characteristic small-angle X-ray scattering pattern for the cuticle. This pattern has been assigned to the cell membrane complex (CMC) between each cuticle scale. Using a simple model of the electron density within the CMC, values have been derived for the average thickness of the beta- and delta-layers which are close to those obtained by electron microscopy. In order to illustrate the potentialities of microdiffraction in studying the properties of the cuticle, the effect of water sorption has been monitored. Using the intensity modelling described above, a 10% swelling of the delta-layer's thickness has been observed. This study shows that structural modifications of the CMC by physical or chemical stress can be followed directly on the cuticle of human hair fibres by microdiffraction analysis.

Profiling lipids across Caucasian and Afro-American hair transverse cuts, using synchrotron infrared microspectrometry.

Synchrotron-based infrared microscopic measurements have been performed on various hair transverse sections, sampled either from the heads of Caucasian or Afro-American subjects. Lipid content of various virgin hair transverse sections was established, with an unprecedented resolution. The variations in shape and intensity of the CH(2), CH(3), amide I and amide II bands, before and after lipid removal by solvent extraction, were profiled, showing clearly that Caucasian hair often contains lipids localized inside the medulla and to a lesser extent inside the cuticle. This statement does not hold for the Afro-American hair analysed. For this, the FT-IR spectra do not change within the hair section and are insensitive to solvent extraction. The importance of the origin of hair on its physical and chemical properties has to be taken into account in future investigations.