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Proteolytic cleavage reveals interaction patterns between silica nanoparticles and two variants of human carbonic anhydrase
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
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2005 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 25, 11903-11906 p.Article 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.

Place, publisher, year, edition, pages
2005. Vol. 21, no 25, 11903-11906 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:liu:diva-50344DOI: 10.1021/la050477uOAI: oai:DiVA.org:liu-50344DiVA: diva2:271240
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2012-11-16
In thesis
1. Conformation of polypeptides at nanoparticles interfaces: protein structural changes and induced folding of peptides
Open this publication in new window or tab >>Conformation of polypeptides at nanoparticles interfaces: protein structural changes and induced folding of peptides
2005 (English)Doctoral 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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2005. 49 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 926
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-24573 (URN)6746 (Local ID)91-852-9749-6 (ISBN)6746 (Archive number)6746 (OAI)
Public defence
2005-03-11, Planck, Fysikhuset, Linköpings Universitet, Linköping, 09:15 (Swedish)
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-11-16

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Lundqvist, MartinAndrésen, CeciliaKarlsson, MartinJonsson, Bengt-Harald

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