Cristiano L. P. Oliveira

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.

Correlation of the Physicochemical and Structural Properties of pDNA/Cationic Liposome Complexes with Their in Vitro Transfection

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R(±)) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R(±) = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R(±) is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R(±) to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R(±) value. Interestingly, for R(±) = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R(±)) in the system. This information is used to explain the transfection differences observed at an R(±) = 9 as compared to R(±) = 3 and 6. Close to the isoneutrality region (R(±) = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.

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.

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.

Dose determination by Monte Carlo — a useful tool in gamma radiation process

Abstract This paper presents results from absorbed dose calculations carried out using the Monte Carlo N-particles (MCNP) code in the Portuguese Gamma Irradiation Facility. The computer running time was chosen in such a way that the statistical errors were always less than 5%. Dose measurements using Amber Perspex and ceric-cerous dosimeters were performed in order to validate the calculations. Simulated and experimental data show good agreement even at positions with a high gradient dose. This fact indicates that the model is reliable. Consequently: (a) the code was used in a correct way; (b) source and geometry of the installation were well defined; and (c) dimension, composition and density of the product were correctly established. The amount of experimental work with dosimeters can thus be reduced. The results show that Monte Carlo simulations can be used as a predictive tool of dose measurements in an irradiation plant. However, some validation measurements must be performed.

Solution Studies and Structural Model of the Extracellular Domain of the Human Amyloid Precursor Protein

The amyloid precursor protein (APP) is the precursor of the beta-amyloid peptide (Abeta), which is centrally related to the genesis of Alzheimers disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89% of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 A calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 A and 130 A, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30% of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed.

A Monte Carlo Study of the Influence of the Geometry Arrangements and Structural Materials on a PGNAA System Performance for Cement Raw Material Analysis

Abstract The MCNP-4A code, running on a Pentium PC, was used to simulate a conveyor belt system for on-line cement raw material analysis, namely the determination of calcium, silicon, iron and aluminium contents. The influence of different system and sample parameters (overall system geometry, neutron moderator/reflector composition and thickness, neutron absorber lining of detector shields, sample bulk density and water content) on the background count rate originating from structural materials was studied. A configuration has been obtained which corresponds to a lower background due to structural materials and a relative higher contribution from γ-rays produced in (n, γ) reactions in the bulk raw material. In this configuration, the dependence of the count rate on the sample density and water content is different in the lower and upper detectors. In the upper detectors, the count rate from inelastic reactions in lead surrounding the neutron source is shown to decrease exponentially with increasing density values, thus enabling density measurements to be made.

Monte Carlo studies of the irradiator geometry of the Portuguese Gamma Irradiation Facility

Abstract This paper describes a complete Monte Carlo study of the irradiator geometry of the Portuguese Gamma Irradiation Facility, using the MCNP code. In this work, eight different source rack configurations were simulated. Routine and non-routine irradiations have been studied. For routine irradiations, both the dose per source activity and the dose uniformity remain constant for all source configurations studied. For non-routine irradiations some dependence on source configuration has been found concerning the dose uniformity in the inner rows.

Nanostructure and giant Hall effect in TMx(SiO2)1−x (TM=Co,Fe,Ni) granular system

Granular TMx(SiO2)1−x (TM=Co,Fe,Ni) thin films were thermally treated at different temperatures and their magnetotransport and structural properties were studied. Hall resistivity decreases with thermal annealing. Structure was analyzed based on small angle x-ray scattering results. A model of polydisperse system of hard spheres was used for obtaining structural parameters. Analysis reveals that a volume fraction of transition-metal atoms (less than 29%) are forming nanospheres. Changes in giant Hall effect upon annealing can depend on a particular combination of nanoparticle diameter, interparticle distance, and size distribution.