1,720,960 research outputs found
Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana
No full textPlants contain a large family of so-called calmodulin-like proteins (CMLs) which differ from canonical calmodulin in that they show greater variability in sequence, length, and number of EF-hand domains. The presence of this extended CML family has raised questions regarding the role of these proteins: are they functionally redundant or do they play specific functions in physiological plant processes? To answer these questions, comprehensive biochemical and structural information on CML proteins is fundamental. Among the 50 CMLs from Arabidopsis thaliana, herein we described the ability of CML7 to bind metal ions focusing on the Ca2+ and Mg2+ sensing properties, as well as on metal-induced conformational changes. Circular dichroism and nuclear magnetic resonance (NMR) studies indicated that both Ca2+ and Mg2+ stabilize CML7, as reflected in conformational rearrangements in secondary and tertiary structure and in increases in thermal stability of the protein. However, the conformational changes that binding induces differ between the two metal ions, and only Ca2+ binding controls a structural transition that leads to hydrophobic exposure, as suggested by 8-anilino-1-naphthalenesulfonic acid fluorescence. Isothermal titration calorimetry data coupled with NMR experiments revealed the presence of two high affinity Ca2+-binding sites in the C-lobe of CML7 and two weaker sites in the N-lobe. The paired nature of these CML7 EF-hands enables them to bind Ca2+ with positive cooperativity within each globular domain. Our results clearly place CML7 in the category of Ca2+ sensors. Along with this, the protein can bind to a model target peptide (melittin) in a Ca2+-dependent manner
A Method for Metal/Protein Stoichiometry Determination Using Thin-Film Energy Dispersive X-ray Fluorescence Spectroscopy
Full text linkA convenient approach is proposed for the quantitation of elemental cofactors in proteins and the determination of metal/protein stoichiometry, on the basis of energy dispersive X-ray fluorescence spectroscopy (EDXRF). The analysis of proteins containing the metals Cu, Fe, Zn, and Ca and also the nonmetallic element P is shown as a demonstration of the generality of the method. In general, the reported method gives limit of detection (LOD) and limit of quantification (LOQ) values in the low ppm range and requires only a few microliters of protein sample at micromolar concentrations. Moreover, sample preparation does not require any digestion steps before the analysis. The expected metal/protein stoichiometry was observed for each protein analyzed, highlighting the precision and accuracy of the method in all the tested cases. Furthermore, it is shown that the method is compatible with multimeric proteins and those with post-translational modifications such as glycosylation
Binding of calcium and target peptide to calmodulin-like protein CML19, the centrin 2 of Arabidopsis thaliana
No full textCalmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca2+-specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Plant calmodulin-like proteins: calcium binding and target interactions
Full text linkCalcium (Ca2+) is an ubiquitous key second messenger in all living organisms, from bacteria to differentiated neuronal cells and is involved in a large variety of biological processes such as apoptosis, cell differentiation and proliferation, fertilization, transcription factors activation and many others. Plants in particular are known to be capable of exploiting Ca2+ signalling in a more sophisticated way than other organisms as demonstrated by the larger set of Ca2+-binding proteins (CBPs). Although the study of CBP has been an open and highly studied field for over a century, a biochemical and structural characterization of many CBPs from different organisms is still missing. Here, we provided a deep characterisation of two plant (Arabidopsis thaliana) Ca2+ sensors, a specific class of CBPs, to understand the differences in their ability to respond to Ca2+ and the regulation of downstream targets. Thus we analysed the metal and target binding properties of Calmodulin like protein 7 and 19 (CML7 and CML19). By exploiting spectroscopic techniques (NMR and CD) we evaluated the conformational changes induced by metal binding and assigned the specific affinities for each binding site by a combination of calorimetric and spectroscopic techniques (ITC and NMR). To further understand the Ca2+ sensing properties and the ability to interact with target proteins, we analysed the interaction of CML7 and CML19 with synthetic peptides, representing the binding region on physiological or model targets. Because the majority of CBPs are characterised by multiple metal binding sites, clustered affinities and cooperativity events, the determination of Ca2+/CBPs stoichiometry requires time and sample consuming procedures and sophisticated instrumentations (i.e. ITC and NMR). Thus, we report the optimisation of a new method, based on X-ray fluorescence, to overcome the most common limitations of the currently used techniques, allowing the quick estimation of the Ca2+/protein stoichiometry using low amounts of sample. Finally, initial studies on the recombinant production and characterisation of a neuronal family of CBPs are reported. These protein are called NECABs and are characterised by a peculiar domain composition and might be involved in the development of neurodegenerative diseases
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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