RESUMEN
More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 °C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 °C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer ß-strands I and VI of the ß-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817-8829) of amide hydrogen exchange at pH 7 and 25 °C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local ß-strands.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Mutación Puntual , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/metabolismo , Sitios de Unión , Cobre/metabolismo , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Estructura Secundaria de Proteína , Superóxido Dismutasa/metabolismo , Zinc/metabolismoRESUMEN
Lectins have widely been used in structural and functional studies of complex carbohydrates. They usually bind carbohydrates with relatively low affinity, but compensate for this by multivalency. This multivalent nature of lectins can sometimes produce unwanted reactions such as agglutination or precipitation of target glycoproteins, when using them in different biological and analytical assays. The mushroom lectin Aleuria aurantia binds to fucose-containing oligosaccharides. It is composed of two identical subunits, and each subunit contains five binding sites for fucose. In this study, two forms of recombinant AAL were produced using site-directed mutagenesis. A monomeric form of AAL was produced by exchanging Tyr6 with Arg6, and a single-site fragment of AAL was produced by insertion of an NdeI restriction enzyme cleavage site and a stop codon in the coding sequence. The AAL forms were expressed as His-tagged proteins in Escherichia coli and purified by affinity chromatography. Binding properties of the two AAL forms were performed using surface plasmon resonance, enzyme-linked lectin assay analyses and isothermal titration calorimetry. Both the monomeric AAL (mAAL) and the single-site AAL (S2-AAL) forms retained their capacity to bind fucosylated oligosaccharides. However, both constructs exhibited properties that differed from the intact recombinant AAL (rAAL). mAAL showed similar binding affinities to fucosylated oligosaccharides as rAAL, but had less hemagglutinating capacity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and, in contrast to rAAL and mAAL, S2-AAL did not bind to sialylated fuco-oligosaccharides. The study shows that molecular engineering is a highly useful tool for producing lectins with more defined properties such as decreased valency and defined specificities and affinities. Thus, this approach has high potential in developing reliable diagnostic and biological assays for carbohydrate analysis.
Asunto(s)
Lectinas/química , Sitios de Unión , Cromatografía de Afinidad , Fucosa/química , Fucosa/metabolismo , Pruebas de Hemaglutinación , Lectinas/aislamiento & purificación , Resonancia por Plasmón de SuperficieRESUMEN
Evolutionary theory explains a wide range of biological phenomena. Proper understanding of evolutionary mechanisms such as natural selection is therefore an essential goal for biology education. Unfortunately, natural selection has time and again proven difficult to teach and learn, and students' resulting understanding is often characterized by misconceptions. Previous research has often focused on the importance of certain key concepts such as variation, differential survival, and change in population. However, so-called threshold concepts (randomness, probability, spatial scale, and temporal scales) have also been suggested to be important for understanding of natural selection, but there is currently limited knowledge about how students use these concepts. We sought to address this lack of knowledge by collecting responses to three different natural selection items from 247 university students from Sweden and Germany. Content analysis (deductive and inductive coding) and subsequent statistical analysis of their responses showed that they overall use some spatial scale indicators, such as individuals and populations, but less often randomness or probability in their explanations. However, frequencies of use of threshold concepts were affected by the item context (e.g., the biological taxa and trait gain or loss). The results suggest that the impact of threshold concepts, especially randomness and probability, on natural selection understanding should be further explored.
Asunto(s)
Biología , Aprendizaje , Selección Genética , Biología/educación , Comprensión , Humanos , Conocimiento , EstudiantesRESUMEN
Extracellular superoxide dismutase (ECSOD) interacts with heparin through its C-terminal domain. In this study we used isothermal titration calorimetry (ITC) to get detailed thermodynamic information about the interaction. We have shown that the interaction between ECSOD and intestinal mucosal heparin (M(w) 6000-30000 Da) is exothermic and driven by enthalpy at physiological salt concentration. However, the contribution from entropy is favorable for binding of small isolated heparin fragments. By studying different size-defined heparin fragments, we also concluded that a hexasaccharide moiety is sufficient for strong binding to ECSOD. The binding involves proton transfer from the buffer to the ECSOD-heparin complex, and the results indicate that the number of ionic interactions made between ECSOD and heparin upon binding varies from three to five for heparin and an octasaccharide fragment, respectively. Surprisingly and despite the many charges found on both the protein and the polysaccharide, our results indicate that the nonionic contribution to the binding is large. From the temperature dependence we have calculated the constant pressure heat capacity change (DeltaC(p)) of the interaction to -644 J K(-1) mol(-1) and -306 J K(-1) mol(-1) for heparin and an octasaccharide, respectively.
Asunto(s)
Heparina/química , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Tampones (Química) , Calorimetría/métodos , Heparina/aislamiento & purificación , Heparina/metabolismo , Humanos , Mucosa Intestinal/química , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Cloruro de Sodio/farmacología , Superóxido Dismutasa/metabolismo , Porcinos , TermodinámicaRESUMEN
More than 100 distinct mutations in the gene (SOD1) for human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS). Studies of these mutant proteins, which often have been performed under far from physiological conditions, have indicated effects on protein stabilities, catalytic activity, and metal binding affinities but with no common pattern. Also, with the knowledge that ALS is a late onset disease it is apparent that protein interactions which contribute to the disorder might, in the natural cellular milieu, depend on a delicate balance between intrinsic protein properties. In this study, we have used experimental conditions as near as possible to the in vivo conditions to reduce artifacts emanating from the experimental setup. Using 1H-15N HSQC NMR spectroscopy, we have analyzed hydrogen exchange at the amide groups of wild-type (wt) CuZnSOD and the fALS-associated G93A SOD variant in their fully metalated states. From analyses of the exchange pattern, we have characterized the local dynamics at 64% of all positions in detail in both the wt and G93A protein. The results show that the G93A mutation had no effect on the dynamics at a majority of the investigated positions. However, the mutation results in local destabilization at the site of the mutation and also in stabilization at a few positions that were apparently scattered over the entire protein surface. Most remarkably, the mutation selectively destabilized the remote metal binding region. The results indicate that the metal binding region may affect the intermolecular protein-protein interactions which cause formation of protein aggregates.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Cobre/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Zinc/metabolismo , Alanina/genética , Cobre/química , Glicina/genética , Humanos , Unión Proteica/genética , Estabilidad Proteica , Zinc/químicaRESUMEN
Science communication is facing a paradigm shift, based on the convergence of exploratory and explanatory visualization. In this article, we coin the term exploranation to denote the way in which visualization methods from scientific exploration can be used to communicate results and how methods in explanatory visualization can enrich exploration.
RESUMEN
Self-assembly is the fundamental but counterintuitive principle that explains how ordered biomolecular complexes form spontaneously in the cell. This study investigated the impact of using two external representations of virus self-assembly, an interactive tangible three-dimensional model and a static two-dimensional image, on student learning about the process of self-assembly in a group exercise. A conceptual analysis of self-assembly into a set of facets was performed to support study design and analysis. Written responses were collected in a pretest/posttest experimental design with 32 Swedish university students. A quantitative analysis of close-ended items indicated that the students improved their scores between pretest and posttest, with no significant difference between the conditions (tangible model/image). A qualitative analysis of an open-ended item indicated students were unfamiliar with self-assembly prior to the study. Students in the tangible model condition used the facets of self-assembly in their open-ended posttest responses more frequently than students in the image condition. In particular, it appears that the dynamic properties of the tangible model may support student understanding of self-assembly in terms of the random and reversible nature of molecular interactions. A tentative difference was observed in response complexity, with more multifaceted responses in the tangible model condition.
Asunto(s)
Bioquímica/educación , Aprendizaje , Sustancias Macromoleculares/química , Estudiantes , Evaluación Educacional , Estudiantes/psicología , Suecia , Enseñanza , Temperatura , EscrituraRESUMEN
Molecular life science is one of the fastest-growing fields of scientific and technical innovation, and biotechnology has profound effects on many aspects of daily life-often with deep, ethical dimensions. At the same time, the content is inherently complex, highly abstract, and deeply rooted in diverse disciplines ranging from "pure sciences," such as math, chemistry, and physics, through "applied sciences," such as medicine and agriculture, to subjects that are traditionally within the remit of humanities, notably philosophy and ethics. Together, these features pose diverse, important, and exciting challenges for tomorrow's teachers and educational establishments. With backgrounds in molecular life science research and secondary life science teaching, we (Tibell and Rundgren, respectively) bring different experiences, perspectives, concerns, and awareness of these issues. Taking the nature of the discipline as a starting point, we highlight important facets of molecular life science that are both characteristic of the domain and challenging for learning and education. Of these challenges, we focus most detail on content, reasoning difficulties, and communication issues. We also discuss implications for education research and teaching in the molecular life sciences.
Asunto(s)
Disciplinas de las Ciencias Biológicas/educación , Biología Molecular/educación , Enseñanza/métodos , InvestigaciónRESUMEN
Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.
Asunto(s)
Lectinas/química , Lectinas/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Sitios de Unión , Secuencia de Carbohidratos , Fucosa/química , Fucosa/metabolismo , Pruebas de Hemaglutinación , Humanos , Técnicas In Vitro , Cinética , Lectinas/genética , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Resonancia por Plasmón de Superficie , Triptófano/químicaRESUMEN
More than 100 point mutations of the superoxide scavenger Cu/Zn superoxide dismutase (SOD; EC ) have been associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, these mutations are scattered throughout the protein and provide no clear functional or structural clues to the underlying disease mechanism. Therefore, we undertook to look for folding-related defects by comparing the unfolding behavior of five ALS-associated mutants with distinct structural characteristics: A4V at the interface between the N and C termini, C6F in the hydrophobic core, D90A at the protein surface, and G93A and G93C, which decrease backbone flexibility. With the exception of the disruptive replacements A4V and C6F, the mutations only marginally affect the stability of the native protein, yet all mutants share a pronounced destabilization of the metal-free apo state: the higher the stability loss, the lower the mean survival time for ALS patients carrying the mutation. Thus organism-level pathology may be directly related to the properties of the immature state of a protein rather than to those of the native species.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Mutación , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/etiología , Dicroismo Circular , Humanos , Fosfinas , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.
Asunto(s)
Alanina/química , Heparina/metabolismo , Sefarosa/análogos & derivados , Sefarosa/química , Superóxido Dismutasa/química , Alanina/genética , Sustitución de Aminoácidos , Cromatografía de Afinidad , Dicroismo Circular , Dimerización , Humanos , Mutagénesis , Conformación Proteica , Estructura Terciaria de Proteína , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents. We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents. This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu(2+) and Zn(2+). The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein. The enzymes were purified to homogeneity and active enzymes were obtained in high yield. The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy.