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Conformation of polypeptides at nanoparticles interfaces: protein structural changes and induced folding of peptides
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
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: urn:nbn:se:liu:diva-24573Local ID: 6746ISBN: 91-852-9749-6 (print)OAI: oai:DiVA.org:liu-24573DiVA: diva2:244894
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
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, 10639-10647 p.Article 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: 2012-11-16
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, 5974-5979 p.Article 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: 2012-11-16
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, 10093-10099 p.Article 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: 2012-11-16
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, 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.

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: 2012-11-16
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, 8169-8173 p.Article in journal (Refereed) Published
Abstract [en]

No abstrack available.

Keyword
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: 2012-11-16Bibliographically approved

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