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  • 1.
    Ahl, Ing-Marie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Thermodynamic Characterization of the Interaction between the C-Terminal Domain of Extracellular Superoxide Dismutase and Heparin by Isothermal Titration Calorimetry2009In: BIOCHEMISTRY, ISSN 0006-2960, Vol. 48, no 41, 9932-9940 p.Article in journal (Refereed)
    Abstract [en]

    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 or small isolated heparin fragments. By studying different size-defined heparin fragments, we also concluded that it 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 oil 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 (Delta C-p) of the interaction to -644 J K-1 mol(-1) and -306 J K-1 mol(-1) for heparin and all octasaccharide, respectively

  • 2.
    Ahl, Ing-Marie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Tibell, Lena A. E.
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Analysis of Effects of Mutations in the C-Terminal Domain of Extracellular Superoxide Dismutase by Isothermal Titration Calorimetry and Phage DisplayManuscript (preprint) (Other academic)
    Abstract [en]

    n/a

  • 3.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Carlsson, Mats
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    PINT: a software for integration of peak volumes and extraction of relaxation rates2013In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 56, no 3, 191-202 p.Article in journal (Refereed)
    Abstract [en]

    We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.

  • 4.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Persson, Bengt
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II2004In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 342, no 2, 619-633 p.Article in journal (Refereed)
    Abstract [en]

    Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

  • 5.
    Andersson, Theresa
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Dolphin, Gunnar T.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Nilsson, Jonas W.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors2005In: Chemistry and Biology, ISSN 1074-5521, Vol. 12, no 11, 1245-1252 p.Article in journal (Refereed)
    Abstract [en]

    Several receptors for human carbonic anhydrase II (HCAII) have been prepared by covalently attaching benzenesulfonamide carboxylates via aliphatic aminocarboxylic acid spacers of variable length to the side chain of a lysine residue in a designed 42 residue helix-loop-helix motif. The sulfonamide group binds to the active site zinc ion of human carbonic anhydrase II located in a 15 Å deep cleft. The dissociation constants of the receptor-HCAII complexes were found to be in the range from low micromolar to better than 20 nM, with the lowest affinities found for spacers with less than five methylene groups and the highest affinity found for the spacer with seven methylene groups. The results suggest that the binding is a cooperative event in which both the sulfonamide residue and the helix-loop-helix motif contribute to the overall affinity.

  • 6.
    Andrasko, Jan
    et al.
    GC UV Centre, Kobergsgränd 2, SE-58731 Linkoping, Sweden.
    Lagesson-Andrasko, Ludmila
    GC UV Centre, Kobergsgränd 2, SE-58731 Linkoping, Sweden.
    Dahlén, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Analysis of Explosives by GC-UV2017In: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 62, no 4, 1022-1027 p.Article in journal (Refereed)
    Abstract [en]

    A mixture of explosives was analyzed by gas chromatography (GC) linked to ultraviolet (UV) spectrophotometry that enabled detection in the range of 178-330 nm. The gas-phase UV spectra of 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), ethylene glycol dinitrate (EGDN), glycerine trinitrate (NG, nitroglycerine), triacetone triperoxide (TATP), and pentaerythritol tetranitrate (PETN) were successfully recorded. The most interesting aspect of the current application is that it enabled simultaneous detection of both the target analyte and its decomposition products. At suitable elevated temperatures of the transfer line between the GC instrument and the UV detector, a partial decomposition was accomplished. Detection was made in real time and resulted in overlaid spectra of the mother compound and its decomposition product. Hence, the presented approach added another level to the qualitative identification of the explosives in comparison with traditional methods that relies only on the detection of the target analyte. As expected, the decomposition product of EGDN, NG, and PETN was NO, while TATP degraded to acetone. DNT and TNT did not exhibit any decomposition at the temperatures used.

    The full text will be freely available from 2018-01-10 16:23
  • 7.
    Aronsson, Göran
    et al.
    Department of Biochemistry, Umeå University.
    Brorsson, Ann-Christin
    Department of Biochemistry, Umeå University.
    Sahlman, Lena
    Department of Biochemistry, Umeå University.
    Jonsson, Bengt Harald
    Department of Biochemistry, Umeå University.
    Remarkably slow folding of a small protein.1997In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 411, no 2-3, 359-364 p.Article in journal (Refereed)
    Abstract [en]

    Equilibrium denaturation of the 72 amino acid alpha/beta-protein MerP, by acid, guanidine hydrochloride, or temperature, is fully reversible and follows a two-state model in which only the native and unfolded states are populated. A cis-trans equilibrium around a proline peptide bond causes a heterogeneity of the unfolded state and gives rise to a slow- and a fast folding population. With a rate constant of 1.2 s(-1) for the major fast folding population, which has none of the common intrinsically slow steps, MerP is the slowest folding protein of this small size yet reported.

  • 8.
    Babu Moparthi, Satish
    et al.
    Aix Marseille University, France.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Vincentelli, Renaud
    University of Aix Marseille, France.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wenger, Jerome
    Aix Marseille University, France.
    Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, no 28386Article in journal (Refereed)
    Abstract [en]

    Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of beta-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.

  • 9.
    Babu Moparthi, Satish
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Institut Fresnel, CNRS UMR 7249, Aix-Marseille Université, Marseille, France.
    Sjölander, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Villebeck, Laila
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL2014In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 7, no 1, 1-15 p.Article, review/survey (Refereed)
    Abstract [en]

    The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.

  • 10.
    Basaiawmoit, R V
    et al.
    Aarhus University.
    Oliveira, C L P
    Aarhus University.
    Runager, K
    Aarhus University.
    Sorensen, C S
    Aarhus University.
    Behrens, M A
    Aarhus University.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Kristensen, T
    Aarhus University.
    Klintworth, G K
    Duke University.
    Enghild, J J
    Aarhus University.
    Skov Pedersen, J
    Aarhus University.
    Otzen, D E
    Aarhus University.
    SAXS Models of TGFBIp Reveal a Trimeric Structure and Show That the Overall Shape Is Not Affected by the Arg124His Mutation2011In: JOURNAL OF MOLECULAR BIOLOGY, ISSN 0022-2836, Vol. 408, no 3, 503-513 p.Article in journal (Refereed)
    Abstract [en]

    Human transforming growth factor beta induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple "beads-on-a-string" arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits.

  • 11.
    Berglund, Anders
    et al.
    Department of Chemistry, Umeå University.
    Brorsson, Ann-Christin
    Biochemistry, Umeå University.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Sethson, Ingmar
    Department of Chemistry, Umeå University.
    The equilibrium unfolding of MerP characterized by multivariate analysis of 2D NMR data2005In: Journal of magnetic resonance (San Diego, Calif. 1997: Print), ISSN 1090-7807, Vol. 172, no 1, 24-30 p.Article in journal (Refereed)
    Abstract [en]

    A general problem when analysing NMR spectra that reflect variations in the environment of target molecules is that different resonances are affected to various extents. Often a few resonances that display the largest frequency changes are selected as probes to reflect the examined variation, especially in the case, where the NMR spectra contain numerous resonances. Such a selection is dependent on more or less intuitive judgements and relying on the observed spectral variation being primarily caused by changes in the NMR sample. Second, recording changes observed for a few (albeit significant) resonances is inevitably accompanied by not using all available information in the analysis. Likewise, the commonly used chemical shift mapping (CSM) [Biochemistry 39 (2000) 26, Biochemistry 39 (2000) 12595] constitutes a loss of information since the total variation in the data is not retained in the projection into this single variable. Here, we describe a method for subjecting 2D NMR time-domain data to multivariate analysis and illustrate it with an analysis of multiple NMR experiments recorded at various folding conditions for the protein MerP. The calculated principal components provide an unbiased model of variations in the NMR spectra and they can consequently be processed as NMR data, and all the changes as reflected in the principal components are thereby made available for visual inspection in one single NMR spectrum. This approach is much less laborious than consideration of large numbers of individual spectra, and it greatly increases the interpretative power of the analysis. © 2004 Elsevier Inc. All rights reserved.

  • 12.
    Bivall Persson, Petter
    et al.
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Cooper, Matthew
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Linköping University, Faculty of Health Sciences.
    Ainsworth, Shaaron
    Learning Sciences Research Institute, University of Nottingham, Nottingham, UK.
    Ynnerman, Anders
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Designing and Evaluating a Haptic System for Biomolecular Education2007In: IEEE Virtual Reality Conference, 2007. VR '07. / [ed] Sherman, W; Lin, M; Steed, A, Piscataway, NJ, USA: IEEE , 2007, 171-178 p.Conference paper (Refereed)
    Abstract [en]

    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 system's 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.

  • 13.
    Bivall Persson, Petter
    et al.
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Cooper, Matthew
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, Faculty of Science & Engineering.
    Ynnerman, Anders
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology.
    Tibell, Lena
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Use of Chemical Force Feedback for Multisensory Insights into Ligand Docking2007In: VII European Symposium of The Protein Society: From Proteins to Proteome, 2007, 151-151 p.Conference paper (Refereed)
  • 14.
    Bivall Persson, Petter
    et al.
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, Faculty of Science & Engineering.
    Tibell, Lena
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Cooper, Matthew
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Ynnerman, Anders
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology.
    Evaluating the Effectiveness of Haptic Visualization in Biomolecular Education - Feeling Molecular Specificity in a Docking Task2006In: 12th IOSTE Symposium, Universiti Science Malaysia , 2006, 745-752 p.Conference paper (Refereed)
    Abstract [en]

    Within the molecular life sciences extensive use is made of visual representations, ranging from sketches to advanced computer graphics, often used to convey abstract knowledge that is difficult for the student to grasp. This work evaluates a new visual and haptic (tactile/kinetic) tool for protein docking in an in situ learning situation by combining qualitative and quantitative methods, performing tests and interviews with students; all aiming at a proper inclusion of visualization tools into biomolecular education. Preliminary results indicate time gains, strong positive affective responses and learning gains from the tasks, however the influence of haptics needs further investigation.

  • 15.
    Brorsson, Ann-Christin
    et al.
    Department of Biochemistry, Umeå University.
    Kjellson, Annika
    Department of Biochemistry, Umeå University.
    Aronsson, Göran
    Biopool AB.
    Sethson, Ingmar
    Department of Organic Chemistry, Umeå University.
    Hambraeus, Charlotta
    University of Southern Stockholm.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    The "two-state folder" MerP forms partially unfolded structures that show temperature dependent hydrogen exchange2004In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 340, no 2, 333-344 p.Article in journal (Refereed)
    Abstract [en]

    We have analysed the folding energy landscape of the 72 amino acid protein MerP by monitoring native state hydrogen exchange as a function of temperature in the range of 7-55°C. The temperature dependence of the hydrogen exchange has allowed us to determine ΔG, ΔH and ΔCp values for the conformational processes that permit hydrogen exchange. When studied with the traditional probes, fluorescence and CD, MerP appears to behave as a typical two-state protein, but the results from the hydrogen exchange analysis reveal a much more complex energy landscape. Analysis at the individual amino acid level show that exchange is allowed from an ensemble of partially unfolded structures (i.e. intermediates) in which the stabilities at the amino acid level form a broad distribution throughout the protein. The formation of partially unfolded structures might contribute to the unusually slow folding of MerP. © 2004 Elsevier Ltd. All rights reserved.

  • 16.
    Brorsson, Ann-Christin
    et al.
    Department of Biochemistry, Umeå University.
    Lundqvist, Martin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Sethson, Ingmar
    Department of Organic Chemistry Umeå University.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern2006In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 357, no 5, 1634-1646 p.Article in journal (Refereed)
    Abstract [en]

    We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (ΔGHX) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions. © 2006 Elsevier Ltd. All rights reserved.

  • 17.
    Carlsson, Uno
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M
    Persson, M
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Mårtensson, Lars-Göran
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Andersson, D
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Aggregation is site-specific in carbonic anhydrase and is prevented by GroEL: The interaction leads to a more flexible structure of both the protein substrate and the chaperonin.2000In: Biophysical Journal, ISSN 0006-3495, Vol. 78, no 1, 202Pos- p.Conference paper (Other academic)
  • 18.
    Chilkova, O.
    et al.
    Dept. of Med. Biochem./Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Johansson, E.
    Dept. of Med. Biochem./Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
    The quaternary structure of DNA polymerase e from Saccharomyces cerevisiae2003In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 278, no 16, 14082-14086 p.Article in journal (Refereed)
    Abstract [en]

    DNA polymerase e (Pol e) trom Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol e have been cumbersome due to protease sensitivity and the limited amounts of Pol e in cells. We have developed a protocol for overexpression and purification of Pol e from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol e was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 Å), a molecular mass for Pol e of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol e. Thus, both DNA polymerase d and Pol e are purified as monomeric complexes, in agreement with accumulating evidence that Pol d and Pol e are located on opposite strands of the eukaryotic replication fork.

  • 19.
    Chirica, Laura C.
    et al.
    Department of Chemistry, Biochemistry, Umeå University, Umeå, Sweden.
    Petersson, Christoffer
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Hurtig, Marina
    Department of Odontology, Umeå University, Umeå, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Borén, Thomas
    Department of Odontology, Umeå University, Umeå, Sweden.
    Lindskog, Sven
    Department of Chemistry, Biochemistry, Umeå University, Umeå, Sweden.
    Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori2002In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1601, no 2, 192-199 p.Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori, the causative agent of peptic ulcer disease, expresses two different forms of the zinc-containing enzyme carbonic anhydrase (CA) (α and β), catalyzing the reversible hydration of CO2. Presumably, the high CO2 requirement of H. pylori implies an important role for this enzyme in the bacterial physiology. In this paper, expression of the CAs has been analyzed in three different strains of the bacterium, 26695, J99 and 17.1, and appears to be independent of CO2 concentration in the investigated range (0.1–10%). Presence of the potent and highly specific CA inhibitor, acetazolamide, in the medium does not seem to inhibit bacterial growth at the given sulfonamide concentration. Moreover, the localization and distribution of the α-CA was analyzed by immunonegative staining, while SDS-digested freeze-fracture immunogold labelling was used for the β-form of the enzyme. The latter method has the advantage of allowing assessment of protein localization to distinct cell compartments and membrane structures. The resulting electron microscopy images indicate a localization of the β-CA in the cytosol, on the cytosolic side of the inner membrane and on the outer membrane facing the periplasmic space. The α-enzyme was found attached to the surface of the bacterium.

  • 20.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Protein denaturation and the denatured state2005In: Encyclopedia of Life Sciences, Wiley-Blackwell , 2005Chapter in book (Other (popular science, discussion, etc.))
    Abstract [en]

    Protein denaturation experiments are routinely used to determine protein stability and to elucidate structural and dynamic effects of mutations, cofactors and ligands. Denatured states of proteins have gained wide interest in recent years owing to their fundamental importance in a wide variety of phenomena such as deciphering the protein folding problem and the molecular understanding of many diseases.

  • 21.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Kalman, B.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Pyrene Excimer Fluorescence as a Proximity Probe for Investigation of Residual Structure in the Unfolded State of Human Carbonic Anhydrase II1997In: FEBS Letters, ISSN 0014-5793, Vol. 420, 63-68 p.Article in journal (Refereed)
  • 22.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Malin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Mårtensson, Lars-Göran
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Andersson, D.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Structural mapping of an aggregation nucleation site in a molten-globule intermediate1999In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 274, 32897-32903 p.Article in journal (Refereed)
  • 23.
    Hennig, Janosch
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Helmholtz Zentrum Munchen GmbH, Germany; Technical University of Munich, Germany.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Museth, Anna Katrine
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. CALTECH, CA 91125 USA.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Local Destabilization of the Metal-Binding Region in Human Copper-Zinc Superoxide Dismutase by Remote Mutations Is a Possible Determinant for Progression of ALS2015In: Biochemistry, ISSN 1520-4995, E-ISSN 0006-2960, Vol. 54, no 2, 323-333 p.Article in journal (Refereed)
    Abstract [en]

    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 degrees 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 degrees 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 beta-strands I and VI of the beta-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 degrees 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 beta-strands.

  • 24. Huang, S.
    et al.
    Sjoblom, B.
    Sauer-Eriksson, A.E.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Organization of an efficient carbonic anhydrase: Implications for the mechanism based on structure-function studies of a T199P/C206S mutant2002In: Biochemistry, ISSN 0006-2960, Vol. 41, no 24, 7628-7635 p.Article in journal (Refereed)
    Abstract [en]

    Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)1 reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and ß-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The ß-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.

  • 25.
    Höst, Gunnar
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign2008In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1784, no 5, 811-815 p.Article in journal (Refereed)
    Abstract [en]

    Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occuring esterases. The design was based on a previously developed strategy,[1] in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with kcat/KM = 625 (± 38) M-1s-1. It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with kcat/KM = 101700 (± 4800) M-1s-1, which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable KM values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV),[1] but for V121A/V143A/T200A no KM could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, kcat/KM is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.

  • 26.
    Höst, Gunnar
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains2006In: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, ISSN 1570-9639, Vol. 1764, no 10, 1601-1606 p.Article in journal (Refereed)
    Abstract [en]

    The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is > 500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.

  • 27.
    Höst, Gunnar
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Razkin, Jesus
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Uppsala University, Department of Chemistry, Uppsala, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Combined Enzyme and Substrate Design: Grafting of a Cooperative Two-Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate2007In: ChemBioChem (Print), ISSN 1439-4227, Vol. 8, no 13, 1570-1576 p.Article in journal (Refereed)
    Abstract [en]

    A histidine-based, two-residue reactive site for the catalysis of hydrolysis of designed sulfonamide-containing para-nitrophenyl esters has been engineered into a scaffold protein. A matching substrate was designed to exploit the natural active site of human carbonic anhydrase II (HCAII) for well-defined binding. In this we took advantage of the high affinity between the active site zinc atom and sulfonamides. The ester substrate was designed to position the scissile bond in close proximity to the His64 residue in the scaffold protein. Three potential sites for grafting the catalytic His-His pair were identified, and the corresponding N62H/H64, F131H/V135H and L198H/P202H mutants were constructed. The most efficient variant, F131H/V135H, has a maximum kcat/KM value of approximately 14 000 M-1 s-1, with a kcat value that is increased by a factor of 3 relative to that of the wild-type HCAII, and by a factor of over 13 relative to the H64A mutant. The results show that an esterase can be designed in a stepwise way by a combination of substrate design and grafting of a designed catalytic motif into a well-defined substrate binding site.

  • 28.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Functional Protein-Nanoparticle-Complexes by Peptide Design and Protein Engineering2008In: 10th International Symposium on Biotechnology, Metal Complexes and Catalysis BMC-X,2008, 2008, 103-104 p.Conference paper (Other academic)
    Abstract [en]

      

  • 29.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Nanopartiklar ger peptider liv2007In: Kemivärlden Biotech, ISSN 1650-0725, no 3, 26-28 p.Article in journal (Other (popular science, discussion, etc.))
  • 30.
    Jonsson, Bengt-Harald
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Broo, Klas
    Lundqvist, Martin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Nygren, Patrik
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Design of Functional Peptide-Nanoparticle Complexes with Potential Applications in Targeted Drug Delivery2008In: BITs 6th annual congress of 2008 International drug discovery science and technology,2008, 2008, 142-143 p.Conference paper (Other academic)
  • 31.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Thermal Induction of an Alternatively Folded State in Human IgG-Fc2011In: Biochemistry, ISSN 1520-4995, E-ISSN 0006-2960, Vol. 50, no 6, 981-988 p.Article in journal (Refereed)
    Abstract [en]

    We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.

  • 32.
    Karlsson, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Adsorption of human carbonic anhydrase II variants to silica nanoparticles occur stepwise: binding is followed by successive conformational changes to a molten-globule-like state2000In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 16, no 22, 8470-8479 p.Article in journal (Refereed)
    Abstract [en]

    The surface adsorption behavior of protein variants of the enzyme human carbonic anhydrase II (HCA II) to silica nanoparticles has been investigated. Various destabilized mutants were produced by site-directed mutagenesis of amino acids located in the interior of the protein. The silica particles induced a molten-globule-like state in all of the variants. All protein variants initially adsorbed to the particles, and then underwent conformational rearrangements in a stepwise manner, as indicated by the loss of activity and the subsequent loss of tertiary structure. Activity, CD, and ANS fluorescence measurements showed that a decrease in the global stability of the protein is strongly correlated to increased rates of conformational change following particle adsorption. In contrast to unfolding processes induced by chemical denaturants or heat, in the transition to the molten-globule-like state induced by the silica particles, the active site region unfolds before the majority of the tertiary interactions are broken.

  • 33.
    Kjellsson, A.
    et al.
    Department of Biochemistry, Umeå University, SE-901 87 Umeå, Sweden.
    Sethson, I.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Hydrogen exchange in a large 29 kD protein and characterization of molten globule aggregation by NMR2003In: Biochemistry, ISSN 0006-2960, Vol. 42, no 2, 363-374 p.Article in journal (Refereed)
    Abstract [en]

    The nature of denatured ensembles of the enzyme human carbonic anhydrase (HCA) has been extensively studied by various methods in the past. The protein constitutes an interesting model for folding studies that does not unfold by a simple two-state transition, instead a molten globule intermediate is highly populated at 1.5 M GuHCl. In this work, NMR and H/D exchange studies have been conducted on one of the isozymes, HCA I. The H/D exchange studies, which were enabled by the previously obtained resonance assignment of HCA I, have been used to identify unfolded forms that are accessible from the native state. In addition, the GuHCl-induced unfolded states of HCA I have also been characterized by NMR at GuHCl concentrations in the 0-5 M range. The most important findings in this work are as follows: (1) Amide protons located in the center of the ß-sheet require global unfolding events for efficient H/D exchange. (2) The molten globule and the native state give similar protection against H/D exchange for all of the observable amide protons (i.e., water seems not to efficiently penetrate the interior of the molten globule). (3) At high protein concentrations, the molten globule can form large aggregates, which are not detectable by solution-state NMR methods. (4) The unfolded state (U), present at GuHCl concentrations above 2 M, is composed of an ensemble of conformations having residual structures with different stabilities.

  • 34. Lindgren, M.
    et al.
    Eaton, S.
    Eaton, G
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Electron Spin Echo Decay at a Probe of Aminoxyl Environment in Spin-labeled Mutants of Carbonic Anhydrase1997In: Journal of the Chemical Society. Perkin transactions II, ISSN 0300-9580, 2549-2554 p.Article in journal (Refereed)
  • 35.
    Lundqvist, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Christensson, Sara
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
    Johansson, Sara
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Broo, Klas
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Proteolytic cleavage reveals interaction patterns between silica nanoparticles and two variants of human carbonic anhydrase2005In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 25, 11903-11906 p.Article in journal (Refereed)
    Abstract [en]

    To characterize the sites on the protein surface that are involved in the adsorption to silica nanoparticles and the subsequent rearrangements of the protein/nanoparticle interaction, a novel approach has been used. After incubation of protein with silica nanoparticles for 2 or 16 h, the protein was cleaved with trypsin and the peptide fragments were analyzed with mass spectrometry. The nanoparticle surface area was in 16-fold excess over available protein surface to minimize the probability that the initial binding would be affected by other protein molecules. When the fragment patterns obtained in the presence and absence of silica nanoparticles were compared, we were able to characterize the protein fragments that interact with the surface. This approach has allowed us to identify the initial binding sites on the protein structure and the rearrangement of the binding sites that occur upon prolonged incubation with the surface.

  • 36.
    Lundqvist, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Nygren, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Broo, Klas
    Linköping University, Department of Physics, Chemistry and Biology, Applied Optics . Linköping University, The Institute of Technology.
    Induction of structure and function in a designed peptide upon adsorption on a silica nanoparticle2006In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 45, no 48, 8169-8173 p.Article in journal (Refereed)
    Abstract [en]

    No abstrack available.

  • 37.
    Lundqvist, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Sethson, Ingmar
    Department of Organic Chemistry, Umeå University, Umeå, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    High-Resolution 2D 1H−15N NMR Characterization of Persistent Structural Alterations of Proteins Induced by Interactions with Silica Nanoparticles2005In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 13, 5974-5979 p.Article in journal (Refereed)
    Abstract [en]

    The binding of protein to solid surfaces often induces changes in the structure, and to investigate these matters we have selected two different protein−nanoparticle systems. The first system concerns the enzyme human carbonic anhydrase II which binds essentially irreversibly to the nanoparticles, and the second system concerns human carbonic anhydrase I which alternate between the adsorbed and free state upon interaction with nanoparticles. Application of the TROSY pulse sequence has allowed high-resolution NMR analysis for both of the protein−nanoparticle systems. For HCAII it was possible to observe spectra of protein when bound to the nanoparticles. The results indicated that HCAII undergoes large rearrangements, forming an ensemble of molten globule-like structures on the surface. The spectra from the HCAI−nanoparticle system are dominated by HCAI molecules in solution. A comparative analysis of variations in intensity from 97 amide resonances in a 1H−15N TROSY spectrum revealed the effects from interaction with nanoparticle on the protein structure at amino acid resolution.

  • 38.
    Lundqvist, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Sethson, Ingmar
    Department of Organic Chemistry, Umeå University, Umeå, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Protein adsorption onto silica nanoparticles: conformational changes depend on the particles' curvature and the protein stability2004In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 20, no 24, 10639-10647 p.Article in journal (Refereed)
    Abstract [en]

    We have analyzed the adsorption of protein to the surfaces of silica nanoparticles with diameters of 6, 9, and 15 nm. The effects upon adsorption on variants of human carbonic anhydrase with differing conformational stabilities have been monitored using methods that give complementary information, i.e., circular dichroism (CD), nuclear magnetic resonance (NMR), analytical ultracentrifugation (AUC), and gel permeation chromatography. Human carbonic anhydrase I (HCAI), which is the most stable of the protein variants, establishes a dynamic equilibrium between bound and unbound protein following mixture with silica particles. Gel permeation and AUC experiments indicate that the residence time of HCAI is on the order of 10 min and slowly increases with time, which allows us to study the effects of the interaction with the solid surface on the protein structure in more detail than would be possible for a process with faster kinetics. The effects on the protein conformation from the interaction have been characterized using CD and NMR measurements. This study shows that differences in particle curvature strongly influence the amount of the protein's secondary structure that is perturbed. Particles with a longer diameter allow formation of larger particle−protein interaction surfaces and cause larger perturbations of the protein's secondary structure upon interaction. In contrast, the effects on the tertiary structure seem to be independent of the particles' curvature.

  • 39.
    Lundqvist, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Sethson, Ingmar
    Department of Organic Chemistry, Umeå University, Umeå, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Transient interaction with nanoparticles "freezes" a protein in an ensemble of metastable near-native conformations2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 30, 10093-10099 p.Article in journal (Refereed)
    Abstract [en]

    It is well-known that adsorption of proteins on interfaces often induces substantial alterations of the protein structure. However, very little is known about whether these conformational changes have any consequence for the protein conformation after desorption from the interface. To investigate this matter, we have selected a protein−particle system in which the enzyme human carbonic anhydrase I (HCAI) alternates between the adsorbed and free state upon interaction with the silica nanoparticles. High-resolution NMR analysis of the protein with the particles present in the sample shows a spectrum that indicates a molten globular-like structure. Removal of particles results in refolding of virtually all HCAI molecules to a fully active form. However, the two-dimensional NMR analysis shows that refolding does not result in a single well-defined protein structure but rather provides an ensemble of protein molecules with near-native conformations. A detailed comparative chemical shift analysis of 108 amide signals in 1H−15N HSQC spectra of native and desorbed HCAI reveals that the most profound effects are located at β-strands in the center of the molecule. The observation of very slow H−D exchange in the central β-strands of HCAI [Kjellsson, A., Sethson, I., and Jonsson, B. H. (2003) Biochemistry 42, 363−374] in conjunction with our results indicates that the kinetic barriers for conformational rearrangements in the central core of the protein are low in the presence of nanoparticles but are very high under native conditions.

  • 40.
    Museth, Anna Katrine
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Brorsson, Anna-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Tibell, Lena A. E.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Selective destabilization of the metal binding region caused by the FALS associated mutation G93A in CuZnSODManuscript (preprint) (Other academic)
    Abstract [en]

    We have, by use of 1H-15N-HSQC NMR spectroscopy, analyzed hydrogen exchange at the amide groups of wtCuZnSOD and the FALS-associated G93A SOD-variant in their fully metallated states. From measurements at near physiological conditions we could analyze the exchange at 64% of all backbone amide groups, which have allowed a detailed characterization of the local dynamics at these positions in both the wt and G93A proteins. 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 mutation and to stabilization at 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 be involved in intermolecular protein-protein interactions, which may constitute the early stages in formation of aggregates.

  • 41.
    Museth, Anna Katrine
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Brorsson, Ann-Christin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology, Visual Information Technology and Applications (VITA). Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    The ALS-Associated Mutation G93A in Human Copper-Zinc Superoxide Dismutase Selectively Destabilizes the Remote Metal Binding Region2009In: BIOCHEMISTRY, ISSN 0006-2960, Vol. 48, no 37, 8817-8829 p.Article in journal (Refereed)
    Abstract [en]

    More than 100 distinct mutations in the gene (SOD 1) 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 oil protein stabilities, catalytic activity, kind 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 H-1-N-15 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.

  • 42.
    Museth, Anna Katrine
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Tibell, Lena A. E.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    The equilibrium between the monomer and dimer form of ALS-related SOD1 mutantsManuscript (preprint) (Other academic)
    Abstract [en]

    No abstract available.

  • 43.
    Nygren, Patrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Broo, Klas
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Fundamental Design Principles That Guide Induction of Helix upon Formation of Stable Peptide−Nanoparticle Complexes2008In: Nano letters (Print), ISSN 1530-6984, Vol. 8, no 7, 1844-1852 p.Article in journal (Refereed)
    Abstract [en]

    We have shown that it is possible to design a peptide that has a very low helical content when free in solution but that adopts a well-defined helix when interacting with silica nanoparticles. From a systematic variation of the amino acid composition and distribution in designed peptides, it has been shown that the ability to form helical structure upon binding to the silica surface is dominated by two factors. First, the helical content is strongly correlated with the net positive charge on the side of the helix that interacts with the silica, and arginine residues are strongly favored over lysine residues in these positions. The second important factor is to have a high net negative charge on the side of the helix that faces the solution. Apparently, both attractive and repulsive electrostatic forces dominate the induction and stabilization of a bound helix. It is also evident that using amino acids that have high propensity to form helix in solution are also advantageous for the formation of helix on surfaces.

  • 44.
    Nygren, Patrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Broo, Klas
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Ederth, Thomas
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Secondary structure in de novo designed peptides induced by electrostatic interaction with particles and membranes.2011Conference paper (Other academic)
  • 45.
    Nygren, Patrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Ederth, Thomas
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Secondary Structure in de Novo Designed Peptides Induced by Electrostatic Interaction with a Lipid Bilayer Membrane2010In: LANGMUIR, ISSN 0743-7463, Vol. 26, no 9, 6437-6448 p.Article in journal (Refereed)
    Abstract [en]

    We show that it is possible to induce a defined secondary structure in de nova designed peptides upon electrostatic attachment to negatively charged lipid bilayer vesicles without partitioning of the peptides into the membrane, and that the secondary structure can be varied via small changes in the primary amino acid sequence of the peptides. The peptides have a random-coil conformation in solution, and results from far-UV circular dichroism spectroscopy demonstrate that the structure induced by the interaction with silica nanoparticles is solely alpha-helical and also strongly pH-dependent. The present study shows that negatively charged vesicles, to which the peptides are electrostatically adsorbed via cationic amino acid residues, induce either alpha-helices or beta-sheets and that the conformation is dependent on both lipid composition and variations in peptide primary structure. The pH-dependence of the vesicle-induced peptide secondary structure is weak, which correlates well with small differences in the vesicles electrophoretic mobility, and thus the surface charge, as the pH is varied.

  • 46.
    Odnell, Anna
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Karshult Municipal Wastewater Treatment, Sweden.
    Recktenwald, Michael
    Kemira Oyj, Finland.
    Stensen, Katarina
    Tekniska Verken Linkoping AB, SE-58278 Linkoping, Sweden; Swedish Meteorol and Hydrol Institute, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. InZymes Biotech AB, Gjuterigatan 1B, SE-58273 Linkoping, Sweden.
    Activity, life time and effect of hydrolytic enzymes for enhanced biogas production from sludge anaerobic digestion2016In: Water Research, ISSN 0043-1354, E-ISSN 1879-2448, Vol. 103, 462-471 p.Article in journal (Refereed)
    Abstract [en]

    As an alternative to energy intensive physical methods, enzymatic treatment of sludge produced at wastewater treatment plants for increased hydrolysis and biogas production was investigated. Several hydrolytic enzymes were assessed with a focus on how enzyme activity and life time was influenced by sludge environments. It could be concluded that the activity life time of added enzymes was limited (amp;lt;24 h) in both waste activated sludge and anaerobic digester sludge environments and that this was, for the majority of enzymes, due to endogenous protease activity. In biogas in situ experiments, subtilisin at a 1% mixture on basis of volatile solids, was the only enzyme providing a significantly increased biomethane production of 37%. However, even at this high concentration, subtilisin could not hydrolyze all available substrate within the life time of the enzyme. Thus, for large scale implementation, enzymes better suited to the sludge environments are needed. (C) 2016 Elsevier Ltd. All rights reserved.

  • 47.
    Olausson, Johan
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Detection of a high affinity binding site in recombinantAleuria aurantia lectin2008In: Glycoconjugate Journal, ISSN 0282-0080, Vol. 25, no 8, 753-762 p.Article in journal (Refereed)
    Abstract [en]

    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 fucosecontaining 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.

  • 48.
    Olausson, Johan
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, The Institute of Technology.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Production and characterization of a monomeric form and a single-site form of Aleuria aurantia lectin2011In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, no 1, 34-44 p.Article in journal (Refereed)
    Abstract [en]

    Lectins have been widely used in structural and functional studies of complex carbohydrates. Lectins usually bind carbohydrates with relatively low affinity but compensate for this by multivalency. When using lectins in different biological and analytical assays the multivalent nature of lectins can sometimes produce unwanted reactions such as agglutination or precipitation of target glycoproteins. The mushroom lectin Aleuria aurantia binds to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. In the present study two forms of recombinant AAL were produced using site-directed mutagenesis. A monomeric form of AAL was produced by exchange of Tyr6 to Arg6, and a monovalent fragment of AAL was produced by insertion of a NdeI restriction enzyme cleavage site and a stop codon in the coding sequence. The AAL forms were expressed as His-tagged proteins in E.coli and purified by affinity chromatography. Binding properties of the two AAL forms were performed using hemagglutination assay, surface plasmon resonance and enzyme-linked lectin assay analyses. Both the monomeric AAL form (mAAL) and the monovalent AAL form (S2-AAL) retained their capacity to bind fucosylated oligosaccharides. However, both constructs exhibited properties that differed from the intact recombinant AAL (rAAL). Monomeric AAL showed similar binding affinities to fucosylated oligosaccharides compared to 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 such as sialyl-Lex. The study shows that molecular engineering techniques may be a tool for producing lectins with more defined properties such as decreased valency and defined specificities and affinities. This may be very valuable for development of reliable diagnostic and biological assays for carbohydrate analysis.

  • 49.
    Owenius, Rikard
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Chemical Physics .
    Jarl, Anngelica
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    An unfolding machine in action: Streching by the chaperonin BroEL of the substrate protin core2006In: Cold Spring Harbor Molecular Chaperones and the heat shock response,2006, 2006Conference paper (Other academic)
  • 50.
    Owenius, Rikard
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jarl, Anngelica
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study2010In: Journal of chemical biology, ISSN 1864-6158, Vol. 3, no 3, 127-39 p.Article in journal (Refereed)
    Abstract [en]

    The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ∼8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.

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