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Protein engineering for biophysical studies of protein folding, stability and surface interactions
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
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 [en]
Biochemistry, protein folding
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28513Local ID: 13663ISBN: 91-85299-76-6 (print)OAI: oai:DiVA.org:liu-28513DiVA: diva2:249323
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
List of papers
1. Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond
Open this publication in new window or tab >>Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond
2002 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 52, 15867-15875 p.Article in journal (Refereed) Published
Abstract [en]

To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten−globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-46774 (URN)10.1021/bi020433+ (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
2. Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations
Open this publication in new window or tab >>Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations
2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 9, 3487-3493 p.Article in journal (Refereed) Published
Abstract [en]

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-45492 (URN)10.1021/bi048610p (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
3. Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase
Open this publication in new window or tab >>Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase
2004 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 21, 6803-6807 p.Article in journal (Refereed) Published
Abstract [en]

The native state of the enzyme human carbonic anhydrase (HCA II) has been stabilized by the introduction of a disulfide bond, the oxidized A23C/L203C mutant. This stabilized protein variant undergoes an apparent two-state unfolding process with suppression of the otherwise stable equilibrium, molten-globule intermediate, which is normally very prone to aggregation. Stopped-flow measurements also showed that lower amounts of the transiently occurring molten globule were formed during refolding. This led to a markedly lowered tendency for aggregation during equilibrium denaturing conditions and, more importantly, to significantly higher reactivation yields upon refolding of the fully denatured protein. Thus, a general strategy to circumvent aggregation during the refolding of proteins could be to stabilize the native state of a protein at the expense of partially folded intermediates, thereby shifting the unfolding behavior from a three-state process to a two-state one.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-45720 (URN)10.1021/bi049709z (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
4. Adsorption of human carbonic anhydrase II variants to silica nanoparticles occur stepwise: binding is followed by successive conformational changes to a molten-globule-like state
Open this publication in new window or tab >>Adsorption of human carbonic anhydrase II variants to silica nanoparticles occur stepwise: binding is followed by successive conformational changes to a molten-globule-like state
2000 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 16, no 22, 8470-8479 p.Article in journal (Refereed) Published
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.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-47929 (URN)10.1021/la0002738 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
5. Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles
Open this publication in new window or tab >>Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles
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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-30369 (URN)10.1529/biophysj.104.054809 (DOI)15916 (Local ID)15916 (Archive number)15916 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13
6. Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability
Open this publication in new window or tab >>Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability
2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 27, 25558-25564 p.Article in journal (Refereed) Published
Abstract [en]

The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation 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. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-50465 (URN)10.1074/jbc.M503665200 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12

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