Lena Tibell

Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: Decreased stability of the apo state

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.

Benefiting from an open-ended experiment? A comparison of attitudes to, and outcomes of, an expository versus an open-inquiry version of the same experiment

In this article we compare outcomes of an open-inquiry and an expository version of a chemistry laboratory experiment at university level for 190 students. The aim of the study was to investigate if these two versions would result in different outcomes depending on the students attitudes towards learning. We used a questionnaire to find out their attitude position prior to the laboratory experiment. The outcome in the different versions of the experiment was evaluated by interviews, questions asked during the experiment and students self-evaluations. The main findings were that the open-inquiry version shows the most positive outcomes regarding learning outcome, preparation time, time spent in the laboratory and student perception of the experiment. The students with low attitude position needed more support to meet the challenge of an open-inquiry experiment, the support being a clearer explanation of the aims, and feedback from the instructor during the experiment.

Educational Challenges of Molecular Life Science: Characteristics and Implications for Education and Research

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 tomorrows 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.

Kinetics and Mechanism of Carbonic Anhydrase Isoenzymes

A mechanism model has been presented that can describe most known kinetic properties of carbonic anhydrase isoenzymes I, II, and III. The essential features of this model include: Nucleophilic attack of metal-bound OH- on CO2 to form metal-bound HCO-3. Formation of metal-bound OH- from metal-bound H2O. In isoenzyme II, and probably also in isoenzyme I, this reaction step involves an intramolecular transfer of H+ between the metal site and a titratable histidine residue via a number of hydrogen-bonded H2O molecules. In isoenzyme II, this step limits the maximal rate of catalysis. Also in isoenzyme III, the H2O-splitting step may be rate limiting, but since this isoenzyme has no titratable active-site histidine, H+ transfer may take place directly with components of the solvent. In isoenzymes I and II, rapid H+ transfer between active site and solution proceeds in a reaction between the titratable histidine residue and buffer molecules. The model can also rationalize a variety of observed inhibition patterns.

Anion Inhibition of CO2 hydration catalyzed by human carbonic anhydrase II: Mechanistic implications

Five monovalent anions, I-, N3-, SCN-, NCO- and Au(CN)2-, were investigated as inhibitors of CO2 hydration catalyzed by human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1). Predominantly uncompetitive inhibition patterns were observed at pH near 9 in all cases. While Dixon plots of Km/V vs. inhibitor concentration were linear, all the investigated anions except NCO- gave nonlinear Dixon plots of 1/V vs. inhibitor concentration. The anion SCN- was also tested at pH 7.4 and 6.4. Essentially noncompetitive patterns of inhibition of CO2 hydration were obtained at both these pH values. These results are analyzed in terms of two rivaling mechanism models, a kinetic scheme originally proposed by Steiner et al. (Steiner, H., Jonsson, B.-H. and Lindskog, S. (1975) Eur. J. Biochem. 59, 253-259) and a rapid-equilibrium kinetic scheme proposed by Pocker and Deits (Pocker, Y. and Deits, T.L. (1982) J. Am. Chem. Soc. 104, 2424-2434). It is concluded that the observed steady-state inhibition patterns are compatible with both models, but that discriminatory data, strongly favouring the model of Steiner et al., are available in the literature.

Designing and Evaluating a Haptic System for Biomolecular Education

In this paper we present an in situ evaluation of a haptic system, with a representative test population, we aim to determine what, if any, benefit haptics can have in a biomolecular education context. We have developed a haptic application for conveying concepts of molecular interactions, specifically in protein-ligand docking. Utilizing a semi-immersive environment with stereo graphics, users are able to manipulate the ligand and feel its interactions in the docking process. The evaluation used cognitive knowledge tests and interviews focused on learning gains. Compared with using time efficiency as the single quality measure this gives a better indication of a systems applicability in an educational environment. Surveys were used to gather opinions and suggestions for improvements. Students do gain from using the application in the learning process but the learning appears to be independent of the addition of haptic feedback. However the addition of force feedback did decrease time requirements and improved the students understanding of the docking process in terms of the forces involved, as is apparent from the students descriptions of the experience. The students also indicated a number of features which could be improved in future development

Determination of the structural role of the N-terminal domain of human extracellular superoxide dismutase by use of protein fusions

The N-terminal domain, containing the 49 N-terminal amino-acid residues, of human extracellular superoxide dismutase (hEC-SOD) has been studied after construction of fusion proteins comprised of the defined domain and human carbonic anhydrase II (HCAII). The specific advantage of this technique is that it allows characterization of properties that are intrinsic to the N-terminal domain of hEC-SOD, i.e., the results are not obscured by properties pertaining to the rest of the hEC-SOD molecule. Moreover, the fusion to HCAII allows a rapid and gentle one-step purification by affinity chromatography. When the N-terminal domain was fused to the N-terminal of HCAII ( = FusNN) a well defined structure was formed and the resulting protein was tetrameric. When the same hEC-SOD-derived domain was fused to the C-terminal of HCAII ( = FusNC), no defined structure of the fused domain could be observed, and the resulting protein was monomeric. It was concluded that a free N-terminus is required for formation of the proper structure of the N-terminal domain.

Exploring relationships between students' interaction and learning with a haptic virtual biomolecular model

This study explores tertiary students interaction with a haptic virtual model representing the specific binding of two biomolecules, a core concept in molecular life science education. Twenty students assigned to a haptics (experimental) or no-haptics (control) condition performed a docking task where users sought the most favourable position between a ligand and protein molecule, while students interactions with the model were logged. Improvement in students understanding of biomolecular binding was previously measured by comparing written responses to a target conceptual question before and after interaction with the model. A log-profiling tool visualized students movement of the ligand molecule during the docking task. Multivariate parallel coordinate analyses explored any relationships in the entire student data set. The haptics group produced a tighter constellation of collected final docked ligand positions in comparison with no-haptics students, coupled to docking profiles that depicted a more fine-tuned ligand traversal. Students in the no-haptics condition employed double the amount of interactive behaviours concerned with switching between different visual chemical representations offered by the model. In the no-haptics group, this visually intense processing was synonymous with erroneously fitting the ligand closer distances to the protein surface. Students who showed higher learning gains tended to engage fewer visual representational switches, and were from the haptics group, while students with a higher spatial ability also engaged fewer visual representational switches, irrespective of assigned condition. From an information-processing standpoint, visual and haptic coordination may offload the visual pathway by placing less strain on visual working memory. From an embodied cognition perspective, visual and tactile sensorimotor interactions in the macroworld may provide access to constructing knowledge about submicroscopic phenomena. The results have cognitive and practical implications for the use of multimodal virtual reality technologies in educational contexts.

Detection of a high affinity binding site in recombinant Aleuria aurantia lectin

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 Kd 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 Kd values in the nanomolar range. This site could bind to oligosaccharides with fucose linked α1-2, α1-3 or α1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with α1-6 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.

The ALS-associated mutation G93A in human copper-zinc superoxide dismutase selectively destabilizes the remote metal binding region.

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.