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Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
2005 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, no 5, 3536-3544 p.Article in journal (Refereed) Published
Abstract [en]

Little is known about the direction and specificity of protein adsorption to solid surfaces, a knowledge that is of great importance in many biotechnological applications. To resolve the direction in which a protein with known structure and surface potentials binds to negatively charged silica nanoparticles, fluorescent probes were attached to different areas on the surface of the protein human carbonic anhydrase II. By this approach it was clearly demonstrated that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein. Furthermore, the adsorption direction is strongly pH-dependent. At pH 6.3, a histidine-rich area around position 10 is the dominating adsorption region. At higher pH values, when the histidines in this area are deprotonated, the protein is also adsorbed by a region close to position 37, which contains several lysines and arginines. Clearly the adsorption is directed by positively charged areas on the protein surface toward the negatively charged silica surface at conditions when specific binding occurs.

Place, publisher, year, edition, pages
2005. Vol. 88, no 5, 3536-3544 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-30369DOI: 10.1529/biophysj.104.054809Local ID: 15916OAI: oai:DiVA.org:liu-30369DiVA: diva2:251191
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13
In thesis
1. Protein engineering for biophysical studies of protein folding, stability and surface interactions
Open this publication in new window or tab >>Protein engineering for biophysical studies of protein folding, stability and surface interactions
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The subject of this thesis can be split into two parts, one that is dealing with the stability and stabilization of proteins on its own merits and a second part that deals with the protein adsorption orientation on solid surfaces and how the stability of a protein influences the behavior at a solid/liquid interface. Thus, the common denominator and focus have been on protein stability. Molecular modeling and site-directed mutagenesis tools have been used to engineer a protein, human carbonic anhydrase II (HCA II), for these studies. The effects of these mutations on stability and surface interactions have then been studied by biophysical methods.

Stabilization of HCA II by an engineered disulfide bridge: To find a way to stabilize the enzyme HCA II, homology modeling against the related and unusually stable carbonic anhydrase from Neisseria gonorrhoeae (NGCA) was performed. We were able to successfully utilize the homology modeling to graft a disulfide bridge from NGCA into the human enzyme. The disulfide bond was not formed spontaneously, but would only form after a prolonged exposure to oxidizing agents. However, formation of the disulfide bridge led to a dramatic stabilization of the native conformation.

Accelerated formation of the disulfide bridge: It was found that it is the conformationally restrained localization of the introduced cysteines that is the reason that the protein is expressed in the reduced state and is not readily oxidized. However, upon exposure to low concentrations of denaturant, corresponding to the lower part of the denaturation curve for the first unfolding transition of the reduced state, there was a striking increase of the oxidation rate of correctly formed disulfide bridges. This provides a method for creating the oxidized disulfide variant of proteins, with engineered cysteines in the interior of proteins, which would otherwise not be formed within an acceptable time span.

Refolding studies of stabilized variant of HCA II: The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition with suppression of the otherwise stable equilibrium. molten-globule intermediate, which normally is very prone to aggregation. Stopped-flow measurements also showed that the population of the transiently occurring molten globule was suppressed during refolding. This circumnavigation of misfolding traps and intermediates led to a markedly lowered tendency for aggregation and to significantly higher reactivation yields upon refolding of the fully denatured protein.

Correlation between protein stability and surface induced denaturation: Negatively charged silica nanoparticles were used in order to determine the influence of protein stability on the denaturation rate upon adsorption. Various destabilized mutants were produced by site-directed mutagenesis of amino acids located in the interior of the protein. The silica nanoparticles induced a molten globule-like state in all of the variants. All protein variants initially adsorbed to the particles, and subsequently underwent conformational rearrangements in a stepwise manner. This study also showed that a decrease in the global stability of the protein is strongly correlated to increased rates of conformational change upon adsorption to the surface.

Determination of protein adsorption orientation: By site-directed labeling fluorescent probes were specifically introduced on the surface of HCA II and it was shown, for the first time, that it is possible to specifically determine the orientation of an adsorbed protein in the native state to a surface (silica nanoparticles). By this approach it was possible to clearly demonstrate that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein and, furthermore, that the adsorption direction is strongly pH-dependent.

Reduction of irreversible protein adsorption by protein stabilization: The strong correlation between decreased stability and increased rates of conformational changes of the protein upon adsorption to surfaces initiated yet another surface study. Three variants of HCA IT with lower, the same, and higher stability than the wild-type protein were monitored by surface plasmon resonance upon adsorption to and desorption from surfaces with fundamentally different properties. Regardless of the nature of the surface there were correlations between (i) the protein stability and kinetics of adsorption with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability, demonstrating that protein engineering for increased stability can be used to reduce irreversible protein adsorption.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2005. 99 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 959
Keyword
Biochemistry, protein folding
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-28513 (URN)13663 (Local ID)91-85299-76-6 (ISBN)13663 (Archive number)13663 (OAI)
Public defence
2005-06-17, Hörsal Planck, Campus Valla, Linköping, 10:15 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-11-27Bibliographically approved

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