Comparative analysis of ribonuclease P RNA using gene sequences from natural microbial populations reveals tertiary structural elements.

PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used to obtain a large collection of diverse RNase P RNA-encoding genes. An alignment of these sequences was used in a comparative analysis of RNase P RNA secondary and tertiary structure. The new sequences confirm the secondary structure model based on sequences from cultivated organisms (with minor alterations in helices P12 and P18), providing additional support for nearly every base pair. Analysis of sequence covariation using the entire RNase P RNA data set reveals elements of tertiary structure in the RNA; the third nucleotides (underlined) of the GNRA tetraloops L14 and L18 are seen to interact with adjacent Watson-Crick base pairs in helix P8, forming A:G/C or G:A/U base triples. These experiments demonstrate one way in which the enormous diversity of natural microbial populations can be used to elucidate molecular structure through comparative analysis.

Structural characterization and comparative analysis of human and piscine cartilage acidic protein (CRTAC1/CRTAC2)

Dissertação de mestrado, Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Allosteric Transition Induced by Mg2+ Ion in a Transactivator Monitored by SERS

We demonstrate the utility of the surface-enhanced Raman spectroscopy (SERS) to monitor conformational transitions in protein upon ligand binding. The changes in protein's secondary and tertiary structures were monitored using amide and aliphatic/aromatic side chain vibrations. Changes in these bands are suggestive of the stabilization of the secondary and tertiary structure of transcription activator protein C in the presence of Mg2+ ion, whereas the spectral fingerprint remained unaltered in the case of a mutant protein, defective in Mg2+ binding. The importance of the acidic residues in Mg2+ binding, which triggers an overall allosteric transition in the protein, is visualized in the molecular model. The present study thus opens up avenues toward the application of SERS as a potential tool for gaining structural insights into the changes occurring during conformational transitions in proteins.

Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain

Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

pH-dependent protein conformational changes in albumin : gold nanoparticle bioconjugates: A spectroscopic study

The conformational changes of bovine serum albumin (BSA) in the albumin:gold nanoparticle bioconjugates were investigated in detail by various spectroscopic techniques including UV-vis absorption, fluorescence, circular dichroism, and Fourier transform infrared spectroscopies. Our studies suggested that albumin in the bioconjugates that was prepared by the common adsorption method underwent substantial conformational changes at both secondary and tertiary structure levels. BSA was found to adopt a more flexible conformational state on the boundary surface of gold nanoparticles as a result of the conformational changes in the bioconjugates. The conformational changes at pH 3.8, 7.0, and 9.0, which corresponded to different isomeric forms of albumin, were investigated, respectively, to probe the pH effect on the conformational changes of BSA in the bioconjugates. The results showed that the pH of the medium influenced the changes greatly and that fluorescence and circular dichroism studies further indicated that the changes were larger at higher pH.

Increased hydrolysis by Thermomyces lanuginosus lipase for omega-3 fatty acids in the presence of a protic ionic liquid

We report that the hydrolytic performance of Thermomyces lanuginosus lipase, TLL, and its selectivity towards concentrating clinically important omega 3 fatty acids was increased by the addition of a protic ionic liquid, pIL, Triethylammonium mesylate, TeaMs. We show that TeaMs has a structure altering effect on TLL, changing both the secondary and tertiary structure of TLL. The thermal activity of TLL was also significantly enhanced by the addition of TeaMs.

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Irreversible inactivation of interleukin 2 in a pump-based delivery environment.

The physical stability of pharmaceutical proteins in delivery environments is a critical determinant of biological potency and treatment efficacy, and yet it is often taken for granted. We studied both the bioactivity and physical stability of interleukin 2 upon delivery via continuous infusion. We found that the biological activity of the delivered protein was dramatically reduced by approximately 90% after a 24-hr infusion program. Only a portion of these losses could be attributed to direct protein deposition on the delivery surfaces. Analysis of delivered protein by size exclusion chromatography gave no indication of insulin-like, surface-induced aggregation phenomena. Examination of the secondary and tertiary structure of both adsorbed and delivered protein via Fourier-transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated that transient surface association of interleukin 2 with the catheter tubing resulted in profound, irreversible structural changes that were responsible for the majority of the biological activity losses.

Psychometric properties of the Spanish version of the Coping Strategies Inventory (CSI) in older people

The aim of this study was to evaluate the psychometric properties of the Coping Strategies Inventory (CSI) in a sample of 243 people aged 55 to 99 years old. The first order factorial structure was similar to the structure of the original version of the CSI and to the Spanish adaptation with general population. Furthermore, high levels of internal consistency and of convergent validity were observed in the different primary scales. Nevertheless, the problem avoidance scale did not surpass the validity and reliability tests. Due to it we propose to delete two of the items of this scale. On the other hand, in this study we confirmed the secondary and tertiary structure obtained in the original version of the inventory that was neither confirmed in the Spanish adaptation study nor the other studies on the psychometric properties of the Spanish version of the CSI.

The effect of graphene oxide and its oxidized debris on the cure chemistry and interphase structure of epoxy nanocomposites

The influence of graphene oxide (GO) and its surface oxidized debris (OD) on the cure chemistry of an amine cured epoxy resin has been investigated by Fourier Transform Infrared Emission Spectroscopy (FT-IES) and Differential Scanning Calorimetry (DSC). Spectral analysis of IR radiation emitted at the cure temperature from thin films of diglycidyl ether of bisphenol A epoxy resin (DGEBA) and 4,4'-diaminodiphenylmethane (DDM) curing agent with and without GO allowed the cure kinetics of the interphase between the bulk resin and GO to be monitored in real time, by measuring both the consumption of primary (1°) amine and epoxy groups, formation of ether groups as well as computing the profiles for formation of secondary (2°) and tertiary (3°) amines. OD was isolated from as-produced GO (aGO) by a simple autoclave method to give OD-free autoclaved GO (acGO). It has been found that the presence of OD on the GO prevents active sites on GO surfaces fully catalysing and participating in the reaction of DGEBA with DDM, which results in slower reaction and a lower crosslink density of the three-dimensional networks in the aGO-resin interphase compared to the acGO-resin interphase. We also determined that OD itself promoted DGEBA homopolymerization. A DSC study further confirmed that the aGO nanocomposite exhibited lower Tg while acGO nanocomposite showed higher Tg compared to neat resin because of the difference in crosslink densities of the matrix around the different GOs.

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: Implications for the tertiary structure of the receptor and for an n-terminal binding site for HIV-1 gp120

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9/1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7/1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9/1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7/1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Organometallic chemistry of diphosphazanes *1: VIII. Synthesis, spectroscopic studies and crystal structure of fac-[Mo(CO)3(MeCN)Ph2PN(iPr)P(Ph)(DMP)], and its reactions with tertiary phosphines

The reaction of fac-[Mo(CO)3(MeCN)3] with the unsymmetrical diphosphazane Ph2PN(iPr)P(Ph)(DMP) (L) gives the complex fac-[Mo(CO)3(MeCN)(L)] (2) in almost quantitative yield. The structure of the complex has been determined by an X-ray diffraction study. The compound reacts with PR3 (where R = Ph, OPh) to give fac-[Mo(CO)3(PR3)(L)] (3a, 4a), which undergoes an intramolecular isomerization to afford mer-[Mo(CO)3(PR3)(L)] (3b, 4b). Synthesis of cis-[Mo(CO)4(L)] (1) and fac-[MO(CO)3L] (2a) and their spectroscopic data are also reported.

Implications of Hydrobiology and Nutrient dynamics on trophic structure and interactions in Cochin backwaters

In the present thesis entitled” Implications of Hydrobiology and Nutrient dynamics on Trophic structure and Interactions in Cochin backwaters”, an attempt has been made to assess the influence of general hydrography, nutrients and other environmental factors on the abundance, distribution and trophic interactions in Cochin backwater system. The study was based on five seasonal sampling campaigns carried out at 15 stations spread along the Cochin backwater system. The thesis is presented in the following 5 chapters. Salient features of each chapter are summarized below: Chapter 1- General Introduction: Provides information on the topic of study, environmental factors, back ground information, the significance, review of literature, aim and scope of the present study and its objectives.Chapter 2- Materials and Methods: This chapter deals with the description of the study area and the methodology adopted for sample collection and analysis. Chapter 3- General Hydrograhy and Sediment Characteristics: Describes the environmental setting of the study area explaining seasonal variation in physicochemical parameters of water column and sediment characteristics. Data on hydrographical parameters, nitrogen fractionation, phosphorus fractionation and biochemical composition of the sediment samples were assessed to evaluate the trophic status. Chapter 4- Nutrient Dynamics on Trophic Structure and Interactions: Describes primary, secondary and tertiary production in Cochin backwater system. Primary production related to cell abundance, diversity of phytoplankton that varies seasonally, concentration of various pigments and primary productivitySecondary production refers to the seasonal abundance of zooplankton especially copepod abundance and tertiary production deals with seasonal fish landings, gut content analysis and proximate composition of dominant fish species. The spatiotemporal variation, interrelationships and trophic interactions were evaluated by statistical methods. Chapter 5- Summary: The results and findings of the study are summarized in the fifth chapter of the thesis.

Secondary bonding in para-substituted diphenyltellurium dichlorides (p-XC6H4)2TeCl2 (X = H, Me, MeO) probed by 125Te MAS NMR spectroscopy. Crystal and molecular structure of (p-MeC6H4)2TeCl2

The solid-state structures of the previously known para-substituted diphenyltellurium dichlorides, (p-XC₆H₄)₂TeCl₂ (X=H (1), Me (2), MeO (3)) were investigated by ¹²⁵Te MAS NMR spectroscopy and in case of 2 by single crystal X-ray diffraction. The ¹²⁵Te-NMR shielding anisotropy (SA) was studied by tensor analyses based on relative intensities of the observed spinning sidebands. Solid-state NMR parameters, namely the isotropic chemical shift (δ_iso), anisotropy (ζ) and asymmetry (η), were discussed in relation to the molecular structures established by X-ray crystallography. The asymmetry (η) was found to be particularly sensitive to structural differences stemming mostly from the diverse secondary Te...Cl interactions, but no correlation with geometric parameters could be established.

Enseñanza media, estructura social y desarrollo en América Latina = Secondary education, social structure and development in Latin America

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