Iris L. Torriani

Poly(phenylsilsesquioxane)s : Structural and morphological characterization

Poly(phenylsilsesquioxane) (PPSQ) polymers that were obtained from different synthetic routes were comparatively studied. The polymers were characterized by infrared and solid-state 29Si NMR spectroscopies. According to the results of X-ray diffraction and thermogravimetric analyses, the materials richest in silanol showed a less organized network and lower weight loss temperature. The morphology of the products was influenced by the preparation conditions. PPSQ, with a morphology rich in spherical particles, was achieved with an n-hexadecyltrimethylammonium bromide template in the reaction medium, whereas the morphology of this polymer obtained in the absence of the template was featureless. Small-angle X-ray scattering analyses revealed that the PPSQ samples showed a predominance of surface-fractal behavior.

Two new sealed sample cells for small angle x-ray scattering from macromolecules in solution and complex fluids using synchrotron radiation

Two different vacuum tight sample cells for in situ temperature dependent small angle scattering from liquids are presented in this article. In the first one, the sample fills a 1 mm thickness gap sealed on both sides by two thin parallel mica windows (volume 300 μl). In the second one, the liquid is injected into a 1 mm cylindrical capillary tube (volume 130 μl). The cells are lodged into temperature controlled chambers directly connected to the beamline vacuum path. Several important improvements with respect to similar instrumentation previously reported are: (1) versatile application of the mica cell, that can be used for all types of samples (gels, liquid crystals, and dispersions in organic solvents) and (2) the design of the chamber for the capillary cell allows registration of wider angle data and a convenient replacement of the capillary tube after each experiment. Signal to background ratio and data reproducibility were tested using protein solutions. We give a brief report of scattering experim...

Polycyclic silicone membranes. Synthesis, characterization and permeability evaluation

In this work, membranes based on polycyclic silicone networks were synthesized from different cyclic siloxanes, through hydrosilylation reactions. These membranes showed good thermal stability, glass transition higher than conventional silicone networks and the absence of pores and phase segregation, with roughness surface. The nanomorphology of the materials was studied by small angle X-ray scattering. Some samples produced practically no scattering and some produced patterns that are typical of non-particulate systems. The gas permeability behavior of the membranes was investigated, showing permeability coefficients higher than those observed for some organic polymers, and selectivity coefficients higher than those found for PDMS based membranes.

The Pyrococcus Exosome Complex STRUCTURAL AND FUNCTIONAL CHARACTERIZATION

The exosome is a conserved eukaryotic enzymatic complex that plays an essential role in many pathways of RNA processing and degradation. Here, we describe the structural characterization of the predicted archaeal exosome in solution using small angle x-ray scattering. The structure model calculated from the small angle x-ray scattering pattern provides an indication of the existence of a disk-shaped structure, corresponding to the “RNases PH ring” complex formed by the proteins aRrp41 and aRrp42. The RNases PH ring complex corresponds to the core of the exosome, binds RNA, and has phosphorolytic and polymerization activities. Three additional molecules of the RNA-binding protein aRrp4 are attached to the core as extended and flexible arms that may direct the substrates to the active sites of the exosome. In the presence of aRrp4, the activity of the core complex is enhanced, suggesting a regulatory role for this protein. The results shown here also indicate the participation of the exosome in RNA metabolism in Archaea, as was established in Eukarya.

A Multitechnique Study of Structure and Dynamics of Polyfluorene Cast Films and the Influence on Their Photoluminescence

This article describes the microstructure and dynamics in the solid state of polyfluorene-based polymers, poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), a semicrystalline polymer, and poly[(9,9-dioctyl-2,7-divinylene-fluorenylene)-alt-co-{2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene vinylene}, a copolymer with mesomorphic phase properties. These structures were determined by wide-angle X-ray scattering (WAXS) measurements. Assuming a packing model for the copolymer structure, where the planes of the phenyl rings are stacked and separated by an average distance of approximately 4.5 A and laterally spaced by about approximately 16 A, we followed the evolution of these distances as a function of temperature using WAXS and associated the changes observed to the polymer relaxation processes identified by dynamical mechanical thermal analysis. Specific molecular motions were studied by solid-state nuclear magnetic resonance. The onset of the side-chain motion at about 213 K (beta-relaxation) produced a small increase in the lateral spacing and in the stacking distance of the phenyl rings in the aggregated structures. Besides, at about 383 K (alpha-relaxation) there occurs a significant increase in the amplitude of the torsion motion in the backbone, producing a greater increase in the stacking distance of the phenyl rings. Similar results were observed in the semicrystalline phase of PFO, but in this case the presence of the crystalline structure affects considerably the overall dynamics, which tends to be more hindered. Put together, our data explain many features of the temperature dependence of the photoluminescence of these two polymers.

Conserved central domains control the quaternary structure of Type I and Type II Hsp40 molecular chaperones

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.

Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution

The fasciculation and elongation protein Zeta 1 (FEZ1) is the mammalian orthologue of the Caenorhabditis elegans protein UNC‐76, which is necessary for axon growth. Human FEZ1 interacts with Protein Kinase C (PKC) and several regulatory proteins involved in functions ranging from microtubule associated transport to transcriptional regulation. Theoretical prediction, circular dichroism, fluorescence spectroscopy, and limited proteolysis of recombinant FEZ1 suggest that it contains disordered regions, especially in its N‐terminal region, and that it may belong to the group of natively unfolded proteins. Small angle X‐ray scattering experiments indicated a mainly disordered conformation, proved that FEZ1 is a dimer of elongated shape and provided overall dimensional parameters for the protein. In vitro pull down experiments confirmed these results and demonstrated that dimerization involves the N‐terminus. Ab‐initio 3D low resolution models of the full‐length conformation of the dimeric constructs 6xHis‐FEZ1(1‐392) and 6xHis‐FEZ1(1‐227) were obtained. Furthermore, we performed in vitro phosphorylation assays of FEZ1 with PKC. The phosphorylation occurred mainly in its C‐terminal region, and does not cause any significant conformational changes, but nonetheless inhibited its interaction with the FEZ1 interacting domain of the protein CLASP2 in vitro. The C terminus of FEZ1 has been reported to bind to several interacting proteins. This suggests that FEZ1 binding and transport function of interacting proteins may be subject to regulation by phosphorylation. Proteins 2009.

Folding and Stability of the Extracellular Domain of the Human Amyloid Precursor Protein

The β-amyloid peptide (Aβ), the major component of the senile plaques found in the brains of Alzheimers disease patients, is derived from proteolytic processing of a transmembrane glycoprotein known as the amyloid precursor protein (APP). Human APP exists in various isoforms, of which the major ones contain 695, 751, and 770 amino acids. Proteolytic cleavage of APP by α- or β-secretases releases the extracellular soluble fragments sAPPα or sAPPβ, respectively. Despite the fact that sAPPα plays important roles in both physiological and pathological processes in the brain, very little is known about its structure and stability. We have recently presented a structural model of sAPPα695 obtained from small-angle x-ray scattering measurements (Gralle, M., Botelho, M. M., Oliveira, C. L. P., Torriani, I., and Ferreira, S. T. (2002) Biophys. J. 83, 3513–3524). We now report studies on the folding and stabilities of sAPPα695 and sAPPα770. The combined use of intrinsic fluorescence, 4–4′-Dianilino-1,1′binaphthyl-5,5′-disulfonic acid (bis-ANS) fluorescence, circular dichroism, differential ultraviolet absorption, and small-angle x-ray scattering measurements of the equilibrium unfolding of sAPPα695 and sAPPα770 by GdnHCl and urea revealed multistep folding pathways for both sAPPα isoforms. Such stepwise folding processes may be related to the identification of distinct structural domains in the three-dimensional model of sAPPα. Furthermore, the relatively low stability of the native state of sAPPα suggests that conformational plasticity may play a role in allowing APP to interact with a number of distinct physiological ligands.

Small Angle X-Ray Scattering from Lipid-Bound Myelin Basic Protein in Solution

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.

Antimony(v) oxide grafted onto a silica gel surface: acidic properties and thermal stability

Antimony(v) oxide grafted onto a silica gel surface was obtained in highly dispersed form, with antimony density of 0.65 atoms nm–2. The material presents Bronsted and Lewis acid properties different from pyrochlore, Sb2O5. These sites disappear upon thermal treatment at 500 °C owing to reaction of antimony oxide with the silica surface.