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  • 1.
    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, E-ISSN 1089-8638, Vol. 342, no 2, p. 619-633Article 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.

  • 2.
    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, E-ISSN 1879-1301, Vol. 12, no 11, p. 1245-1252Article 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.

  • 3.
    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, E-ISSN 1089-8638, Vol. 357, no 5, p. 1634-1646Article 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.

  • 4.
    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, p. 142-143Conference paper (Other academic)
  • 5.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Conformation of polypeptides at nanoparticles interfaces: protein structural changes and induced folding of peptides2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis can be divided into two parts. The first deals with characterization of structural and dynamic consequences for proteins of interactions with a solid surface in the form of silica nanoparticles. The studies were conducted on isoenzymes and mutant variants of Human Carbonic Anhydrase that have virtually identical topology but differ considerably with respect to stability. The nanoparticles were chosen as the solid phase because their small sizes allow the use of spectroscopic techniques that are usually employed to study molecular interactions in solution.

    CD measurements of the interactions between HCAI and nanoparticles with different diameters show that the perturbation of the secondary structure is dependent on the curvature of the nanoparticle. A relatively flat surface causes greater perturbation because it allows a larger interaction area with the. protein than a curved surface. Use of the TROSY pulse sequence allowed NMR spectra of a large stable complex of HCAII and solid nanoparticles to be recorded. The NMR study confirmed earlier conclusions based on CD measurements that HCAII undergoes major structural rearrangements upon binding to the nanoparticles and that the protein continues to rearrange for at least two weeks after binding. Sedimentation equilibrium AUC and gel permeation chromatography experiments established that HCAI is in a true equilibrium between forms that are free and forms that are bound to the nanoparticles for at least seven days. NMR studies of HCAI-nanoparticle systems showed that residues in the central ß-strands of HCAI do not regain their native conformation during the time they are dissociated from the nanoparticles i.e. information about the bound state were gleaned from studies of free molecules. Further NMR studies showed that the perturbations persist for long time even after removal of the nanoparticles from the solution. Surprisingly, the conformational heterogeneity did not disturb the delicate positioning of the active site residues that is required for full catalytic activity. A novel approach was used to characterize the initial binding of HCAII to the nanoparticles and subsequent structural alterations of the protein. MALDI-TOP mass spectrometry was used to analyse the fragment patterns after proteolytic cleavage in the presence of nanoparticles and the results were compared with corresponding fragment pattern for a native sample. The initial binding site of HCAII was shown to include parts of the N- and C-termini and the major subsequent structural rearrangements were also characterized.

    The second part of this thesis concerns an approach to use surface interaction to regulate the structure and function of designed peptides. Using de novo design, a peptide was constructed that would be unstructured in solution, but would be "forced" to adopt a well-defmed helical structure following adsorption to silica nanoparticles. Moreover, the design also included precisely placed amino acids that were intended to form a functional catalytic site upon induction of the helix on the surface of the nanoparticles. Characterization of the structure and function of the designed peptide using CD and activity measurements show that the nanoparticles can be used, as intended, to induce structure in the peptide and switch on the catalytic function.

    List of papers
    1. Protein adsorption onto silica nanoparticles: conformational changes depend on the particles' curvature and the protein stability
    Open this publication in new window or tab >>Protein adsorption onto silica nanoparticles: conformational changes depend on the particles' curvature and the protein stability
    2004 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 20, no 24, p. 10639-10647Article in journal (Refereed) Published
    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.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-39672 (URN)10.1021/la0484725 (DOI)50688 (Local ID)50688 (Archive number)50688 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
    2. High-Resolution 2D 1H−15N NMR Characterization of Persistent Structural Alterations of Proteins Induced by Interactions with Silica Nanoparticles
    Open this publication in new window or tab >>High-Resolution 2D 1H−15N NMR Characterization of Persistent Structural Alterations of Proteins Induced by Interactions with Silica Nanoparticles
    2005 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 13, p. 5974-5979Article in journal (Refereed) Published
    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.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-30402 (URN)10.1021/la050569j (DOI)15958 (Local ID)15958 (Archive number)15958 (OAI)
    Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13
    3. Transient interaction with nanoparticles "freezes" a protein in an ensemble of metastable near-native conformations
    Open this publication in new window or tab >>Transient interaction with nanoparticles "freezes" a protein in an ensemble of metastable near-native conformations
    2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 30, p. 10093-10099Article in journal (Refereed) Published
    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.

    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-50441 (URN)10.1021/bi0500067 (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12
    4. Proteolytic cleavage reveals interaction patterns between silica nanoparticles and two variants of human carbonic anhydrase
    Open this publication in new window or tab >>Proteolytic cleavage reveals interaction patterns between silica nanoparticles and two variants of human carbonic anhydrase
    Show others...
    2005 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 25, p. 11903-11906Article in journal (Refereed) Published
    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.

    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-50344 (URN)10.1021/la050477u (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12
    5. Induction of structure and function in a designed peptide upon adsorption on a silica nanoparticle
    Open this publication in new window or tab >>Induction of structure and function in a designed peptide upon adsorption on a silica nanoparticle
    2006 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 45, no 48, p. 8169-8173Article in journal (Refereed) Published
    Abstract [en]

    No abstrack available.

    Keywords
    Amino acids, catalysis, helical structures, nanoparticles, peptides
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-14548 (URN)10.1002/anie.200600965 (DOI)
    Available from: 2008-02-25 Created: 2008-02-25 Last updated: 2017-12-13Bibliographically approved
  • 6.
    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, p. 11903-11906Article 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.

  • 7.
    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, p. 8169-8173Article in journal (Refereed)
    Abstract [en]

    No abstrack available.

  • 8.
    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, p. 5974-5979Article 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.

  • 9.
    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, p. 10639-10647Article 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.

  • 10.
    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, p. 10093-10099Article 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.

  • 11.
    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.

  • 12.
    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, p. 8817-8829Article 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.

  • 13.
    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, E-ISSN 1530-6992, Vol. 8, no 7, p. 1844-1852Article 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.

  • 14.
    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)
  • 15.
    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, p. 6437-6448Article 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.

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