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  • 1.
    Almstedt, Karin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Lundqvist, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Carlsson, Jonas
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Persson, Bengt
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Jonsson, Bengt-Harald
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II2004Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 342, nr 2, s. 619-633Artikel i tidskrift (Refereegranskat)
    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.
    Ammenberg, Jonas
    et al.
    Linköpings universitet, Institutionen för ekonomisk och industriell utveckling, Industriell miljöteknik. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Biogas Research Center.
    Svensson, Bo
    Linköpings universitet, Institutionen för tema, Tema Miljöförändring. Linköpings universitet, Filosofiska fakulteten. Linköpings universitet, Biogas Research Center.
    Karlsson, Magnus
    Linköpings universitet, Institutionen för ekonomisk och industriell utveckling, Energisystem. Linköpings universitet, Biogas Research Center.
    Svensson, Niclas
    Linköpings universitet, Institutionen för ekonomisk och industriell utveckling, Industriell miljöteknik. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Biogas Research Center.
    Björn, Annika
    Linköpings universitet, Institutionen för tema, Tema Miljöförändring. Linköpings universitet, Filosofiska fakulteten. Linköpings universitet, Biogas Research Center.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Biogas Research Center.
    Tonderski, Karin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Biogas Research Center.
    Eklund, Mats
    Linköpings universitet, Institutionen för ekonomisk och industriell utveckling, Industriell miljöteknik. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Biogas Research Center.
    Biogas Research Center, BRC: Slutrapport för etapp 12015Rapport (Övrigt vetenskapligt)
    Abstract [sv]

    Biogas Research Center (BRC) är ett kompetenscentrum för biogasforskning som finansieras av Energimyndigheten, LiU och ett flertal externa organisationer med en tredjedel vardera. BRC har en mycket bred tvärvetenskaplig inriktning och sammanför biogasrelaterad kompetens från flera olika områden för att skapa interaktion på flera olika plan:

    • mellan näringsliv, akademi och samhälle,
    • mellan olika perspektiv, samt
    • mellan olika discipliner och kompetensområden.

    BRC:s vision är:

    Resurseffektiva biogaslösningar finns genomförda i många nya tillämpningar och bidrar till en mer hållbar energiförsörjning, förbättrat miljötillstånd och goda affärer.

    BRC:s särskilda roll för att uppnå denna vision är att bidra med kunskapsförsörjning och process-/teknikutveckling för att facilitera utveckling, innovation och implementering av biogaslösningar. Resurseffektivitet är ett nyckelord, vilket handlar om att förbättra befintliga processer och system samt utveckla biogaslösningar i nya sektorer och möjliggöra användning av nya substrat.

    For BRC:s etapp 1, den första tvåårsperioden mellan 2012-2014, var forskningsprojekten organiserade enligt tabellen nedan. Den visar viktiga utmaningar för biogasproducenter och andra intressenter, samt hur dessa ”angreps” med åtta forskningsprojekt. Fem av projekten var av explorativ karaktär i bemärkelsen att de var bredare och mer framtidsorienterade - exempelvis utvärderade flera möjliga tekniska utvecklingsmöjligheter (EP1-5). Tre projekt hade ett tydligare fokus på teknik- och processutveckling (DP6-8).

    I den här slutrapporten ges en kortfattad bakgrundsbeskrivning och det finns en introduktion till vad den här typen av kompetenscentrum innebär generellt. Därefter finns mer detaljerad information om BRC, exempelvis gäller det centrumets etablering, relevans, vision, hörnstenar och utveckling. De deltagande organisationerna presenteras, både forskargrupperna vid Linköpings universitet och partners och medlemmar. Vidare finns en mer utförlig introduktion till och beskrivning av utmaningarna i tabellen och kortfattat information om forskningsprojekten, följt av ett kapitel som berör måluppfyllelse och den externa utvärdering som gjorts av BRC:s verksamhet. Detaljerad, listad information finns till stor del i bilagorna.

    Kortfattat kan det konstateras att måluppfyllelsen överlag är god. Det är speciellt positivt att så många vetenskapliga artiklar publicerats (eller är på gång att publiceras) kopplat till forskningsprojekten och även i det vidare centrumperspektivet. Helt klart förekommer en omfattande verksamhet inom och kopplat till BRC. I etapp 2 är det viktigt att öka andelen mycket nöjda partner och medlemmar, där nu hälften är nöjda och hälften mycket nöjda. Det handlar framför allt om stärkt kommunikation, interaktion och projektledning. Under 2015 förväntas åtminstone två doktorsexamina, där avhandlingarna har stor koppling till forskningen inom etapp 1.

    I början på år 2014 skedde en extern utvärdering av verksamheten vid BRC med huvudsyftet att bedöma hur väl centrumet lyckats med etableringen samt att granska om det fanns förutsättningar för framtida framgångsrik verksamhet. Generellt var utfallet mycket positivt och utvärderarna konstaterade att BRC på kort tid lyckats etablera en verksamhet som fungerar väl och engagerar det stora flertalet deltagande aktörer, inom relevanta områden och där de flesta involverade ser BRC som en befogad och väl fungerande satsning, som de har för avsikt att även fortsättningsvis stödja. Utvärderingen bidrog också med flera relevant tips och till att belysa utmaningar.

    Utöver denna slutrapport finns separata publikationer från forskningsprojekten.

    Arbetet som presenteras i rapporten har finansierats av Energimyndigheten och de medverkande organisationerna.

  • 3.
    Ek, Anders
    et al.
    Swedish Biogas International Korea Co.
    Hallin, Sara
    Svensk Biogas FoU.
    Vallin, Lina
    Svensk Biogas FoU.
    Schnurer, Anna
    Swedish University of Agricultural Sciences, Uppsala.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Slaughterhouse waste co-digestion - Experiences from 15 years of full-scale operation2011Ingår i: World Renewable Energy Congress - Sweden 8-13 May, 2011: Volume 1 (Bioenergy Technology) / [ed] Bahram Moshfegh, Linköping: Linköping University Electronic Press, 2011, Vol. 009, s. 64-71Konferensbidrag (Refereegranskat)
    Abstract [en]

    At Tekniska Verken in Linköping AB (TVAB) there is a long time experience of handling and producing biogas from large volumes of slaughterhouse waste. Experiences from research and development and plant operations have lead to the implementation of several process improving technological/biological solutions. We can in this paper describe how the improvements have had several positive effects on the process, including energy savings, better odor control, higher gas quality, increased organic loading rates and higher biogas production with maintained process stability. In addition, it is described how much of the process stability in anaerobic digestion of slaughter house waste relates to the plant operation, which allow the microbiological consortia to adapt to the substrate. Since digestion of proteinaceous substrates like slaughterhouse waste lead to high ammonia loads, special requirements in ammonia tolerance are placed on the microbiota of the anaerobic digestion. Biochemical assays revealed that the main route for methane production proceed through syntrophic acetate oxidation, which require longer retention times than methane production by acetoclastic methanogens. Thus, the long retention time of the plant, accomplished by a low dilution of the substrate, is a vital component of the process stability when treating high protein substrates like slaughterhouse waste.

  • 4. Hellman, Jan
    et al.
    Ek, Anders
    Sundberg, Carina
    Linköpings universitet, Institutionen för tema, Tema vatten i natur och samhälle. Linköpings universitet, Filosofiska fakulteten.
    Johansson, Mariana
    Svensson, Bo
    Linköpings universitet, Institutionen för tema, Tema vatten i natur och samhälle. Linköpings universitet, Filosofiska fakulteten.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Mechanisms of increased methane production through re-circulation of magnetic biomass carriers in an experimental continuously stirred tank reactor2010Konferensbidrag (Refereegranskat)
    Abstract [en]

    Magnetite particles were used in a semi-continuous process as magnetic biomass carriers to separate and re-introduce microorganisms in a CSTR reactor. In comparison to a control reactor the methane content during the semi-continuous process was elevated when magnetite particles were used. The difference was most apparent during the fermentative step directly after feeding and upon direct spiking with volatile fatty acids. Total DNA quantification of the separated magnetite particles revealed high association of microorganisms. Furthermore, quantitative real-time PCR analysis of the associated microbial consortia indicated that the hydrogenotrophic Methanobacteriales was overrepresented at the particle surface. Thus, the increased methane production could be coupled to both the crowding and shorter interspecies distances between the groups involved in anaerobic digestion, as well as a preferential adsorption of hydrogenotrophs. By bringing the hydrogenotrophs closer to the primary fermentative bacteria and increasing their relative number the produced hydrogen during acidogenesis is more effectively utilized and more carbon dioxide is converted to methane. Furthermore, by the same cause, the rate of acetogenesis increased as the hydrogenotrophs more effectively could consume the hydrogen produced and thereby keep the hydrogen partial pressure low.

  • 5.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Protein engineering for biophysical studies of protein folding, stability and surface interactions2005Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

    Delarbeten
    1. Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond
    Öppna denna publikation i ny flik eller fönster >>Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond
    2002 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, nr 52, s. 15867-15875Artikel i tidskrift (Refereegranskat) 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.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-46774 (URN)10.1021/bi020433+ (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
    2. Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations
    Öppna denna publikation i ny flik eller fönster >>Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations
    2005 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 9, s. 3487-3493Artikel i tidskrift (Refereegranskat) 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).

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-45492 (URN)10.1021/bi048610p (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
    3. Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase
    Öppna denna publikation i ny flik eller fönster >>Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase
    2004 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 21, s. 6803-6807Artikel i tidskrift (Refereegranskat) 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.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-45720 (URN)10.1021/bi049709z (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 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
    Öppna denna publikation i ny flik eller fönster >>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 (Engelska)Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 16, nr 22, s. 8470-8479Artikel i tidskrift (Refereegranskat) 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.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-47929 (URN)10.1021/la0002738 (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 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
    Öppna denna publikation i ny flik eller fönster >>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 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, nr 5, s. 3536-3544Artikel i tidskrift (Refereegranskat) 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.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-30369 (URN)10.1529/biophysj.104.054809 (DOI)15916 (Lokalt ID)15916 (Arkivnummer)15916 (OAI)
    Tillgänglig från: 2009-10-09 Skapad: 2009-10-09 Senast uppdaterad: 2017-12-13
    6. Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability
    Öppna denna publikation i ny flik eller fönster >>Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability
    2005 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, nr 27, s. 25558-25564Artikel i tidskrift (Refereegranskat) 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.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-50465 (URN)10.1074/jbc.M503665200 (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-12
  • 6.
    Karlsson, Martin
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Adsorption at the liquid-solid Interface - Influence of protein stability on conformational changes2007Ingår i: Encyclopedia of surfaces and colloid science / [ed] Ponisseril Somasundaran, Taylor & Francis, 2007, 2, Vol. 1Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Protein adsorption has large implications in a variety of fields and can be both a problem and an asset. Most often protein adsorption is accompanied by structural changes in the adsorbed protein. The degree and rate of these changes are dependent on the surface, conditions during adsorption and experimental set up as well as of intrinsic properties of the protein. The effect of conformational changes influences both practical applications and experimental results in studies of protein adsorption at the liquid/solid interface. The intrinsic property of the protein that is most instrumental for conformational changes upon adsorption is the stability of the protein. Hence, large efforts have been directed towards analysis of how both the nature of surfaces and conditions influence the stability of proteins upon adsorption. Less work has been focused on the reversed view, i.e. how the stability of proteins influences adsorption, the rate and degree of the subsequent conformational changes as well as the effects of these changes. However, the increasing use of proteins in a variety of medical and biotechnological applications requires a deeper knowledge of the importance and effects of stabilizing interactions in the protein structure. Engineered stabilized proteins that are less affected by surface interactions should be of potential use for various practical purposes.

  • 7.
    Karlsson, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles2005Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, nr 5, s. 3536-3544Artikel i tidskrift (Refereegranskat)
    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.

  • 8.
    Karlsson, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Ekeroth, Johan
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Elwing, Hans
    Department of Cell and Molecular Biology, Göteborg University, Sweden.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability2005Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, nr 27, s. 25558-25564Artikel i tidskrift (Refereegranskat)
    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.

  • 9.
    Karlsson, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Jonsson, Bengt-Harald
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Adsorption of human carbonic anhydrase II variants to silica nanoparticles occur stepwise: binding is followed by successive conformational changes to a molten-globule-like state2000Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 16, nr 22, s. 8470-8479Artikel i tidskrift (Refereegranskat)
    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.

  • 10.
    Karlsson, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Karlsson, Carin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations2005Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 9, s. 3487-3493Artikel i tidskrift (Refereegranskat)
    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).

  • 11.
    Karlsson, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Olofsson, Patrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase2004Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 21, s. 6803-6807Artikel i tidskrift (Refereegranskat)
    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.

  • 12.
    Linhult, M.
    et al.
    Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden.
    Gulich, S.
    Gülich, S., Dept. of Molec. Biophys./Biochem., Yale University, New Haven, CT, United States.
    Graslund, T.
    Gräslund, T., Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden.
    Simon, A.
    Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden.
    Karlsson, Martin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Sjoberg, A.
    Sjöberg, A., Affibody AB, Bromma, Sweden.
    Nord, K.
    Affibody AB, Bromma, Sweden.
    Hober, S.
    Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden, Department of Biotechnology, KTH, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.
    Improving the Tolerance of a Protein A Analogue to Repeated Alkaline Exposures Using a Bypass Mutagenesis Approach2004Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 55, nr 2, s. 407-416Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule. © 2004 Wiley-Liss, Inc.

  • 13.
    Lundqvist, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Andrésen, Cecilia
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    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
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Broo, Klas
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
    Jonsson, Bengt-Harald
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Proteolytic cleavage reveals interaction patterns between silica nanoparticles and two variants of human carbonic anhydrase2005Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, nr 25, s. 11903-11906Artikel i tidskrift (Refereegranskat)
    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.

  • 14.
    Mishra, Rajesh
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Olofsson, Linus
    Karlsson, Martin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Nicholls, Ian A.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    A conformationally isoformic thermophilic protein with high kinetic unfolding barriers2008Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 65, nr 5, s. 827-839Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The basis for the stability of thermophilic proteins is of fundamental interest for extremophile biology. We investigated the folding and unfolding processes of the homotetrameric Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). TBADH subunits were 4.8 kcal/mol less stable towards guanidinium chloride (GdmCl) unfolding compared to urea, indicating ionic modulation of TBADH stability. Strongly denaturing conditions promoted mono-exponential unfolding kinetics with linear dependence on denaturant concentration. Here TBADH unfolded >40-fold slower when extrapolated from urea as compared to GdmCl unfolding. A marked unfolding hysteresis was shown when comparing refolding and unfolding in urea. An unusual biphasic unfolding trajectory with an exceptionally slow phase at intermediate concentrations of GdmCl and urea was also observed. We advocate that TBADH forms two distinctly different tetrameric isoforms, and likely an ensemble of native states. This unusual supramolecular folding behavior has been shown responsible for formation of amyloidotic yeast prion strains and can have functional importance for TBADH. © 2008 Birkhaueser.

  • 15.
    Moparthi, Satish Babu
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Fristedt, Rikard
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Mishra, Rajesh
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Almstedt, Karin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates2010Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, nr 6, s. 1137-1145Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The single-domain cyclophilin 18 (Cyp18) has long been known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and was proposed by us to also function as a chaperone [Freskgård, P.-O., Bergenhem, N., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1992) Science 258, 466−468]. Later several multidomain PPIs were demonstrated to work as both a peptidyl-prolyl cis/trans isomerase and a chaperone. However, the chaperone ability of Cyp18 has been debated. In this work, we add additional results that show that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e., function as both a folding catalyst and a chaperone. Refolding experiments were performed using severely destabilized mutants of human carbonic anhydrase II under conditions where the unfolding reaction is significant and a larger fraction of a more destabilized variant populates molten globule-like intermediates during refolding. A correlation of native state protein stability of the substrate protein versus Cyp18 chaperone activity was demonstrated. The induced correction of misfolded conformations by Cyp18 likely functions through rescue from misfolding of transient molten globule intermediates. ANS binding data suggest that the interaction by Cyp18 leads to an early stage condensation of accessible hydrophobic portions of the misfolding-prone protein substrate during folding. The opposite effect was observed for GroEL known as an unfoldase at early stages of refolding. The chaperone effect of Cyp18 was also demonstrated for citrate synthase, suggesting a general chaperone effect of this PPI.

  • 16.
    Mårtensson, Lars-Göran
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond2002Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, nr 52, s. 15867-15875Artikel i tidskrift (Refereegranskat)
    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.

  • 17.
    Nordell, Erik
    et al.
    Linköpings universitet, Biogas Research Center. Tekniska Verken Linkoping AB Publ, Dept Biogas RandD, SE-58115 Linkoping, Sweden .
    B Hansson, Anna
    Linköpings universitet, Biogas Research Center. Tekniska Verken Linkoping AB Publ, Dept Biogas RandD, SE-58115 Linkoping, Sweden .
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Biogas Research Center. Tekniska Verken Linkoping AB Publ, Dept Biogas RandD, SE-58115 Linkoping, Sweden .
    Zeolites relieves inhibitory stress from high concentrations of long chain fatty acids2013Ingår i: Waste Management, ISSN 0956-053X, E-ISSN 1879-2456, Vol. 33, nr 12, s. 2659-2663Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein and fat rich slaughterhouse waste is a very attractive waste stream for the production of biogas because of the high biochemical methane potential of the substrate. The material has however some drawbacks as the sole material for biogas production due to the production of several process disturbing metabolites such as ammonia, sulfides and long chain fatty acids. We can in this work present results that show that zeolites have the potential to relieve inhibitory stress from the presence of long chain fatty acids. Moreover, the results strongly indicate that it is mainly acetic acid consumers that are most negatively affected by long chain fatty acids and that the mechanism of stress relief is an adsorption of long chain fatty acids to the zeolites. In addition to this, it is shown that the effect is immediate and that only a small amount of zeolites is necessary to cancel the inhibitory effect of long chain fatty acids.

  • 18. Nordell, Erik
    et al.
    Hallin, Sara
    Johansson, Mariana
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    The Diverse Response on Degradation Rate of Different Substrates Upon Addition of Zeolites2010Ingår i: 3rd International Symposium on Energy from Biomass and Waste. Venice, Italy, Nov 8-11, 2010., 2010Konferensbidrag (Refereegranskat)
    Abstract [en]

    SUMMARY: This study evaluates the effects of addition of the natural zeolite clinoptilolite to anaerobic digesters treating different materials in both batch and continuous lab-scale setups. Zeolite addition to a CSTR with high ammonium levels (5 g NH4+/L) was performed with the purpose to investigate the effects on the amount of free ammonium. We also investigated non ammonium related effects of zeolite addition in dosage between 0-10 g zeolite/L to distinguish any beneficial properties effects. The concentration that was found to be most suitable for slaughterhouse waste (5 g zeolite/L) was subsequently tested and compared in batch conditions for slaughterhouse waste, thin stillage from ethanol production and sewage sludge. Batch experiments with slaughterhouse waste with 5 g zeolite/L significantly decreased the lag phase and in effect increased the degradation rate; no similar effect could be identified for the other substrates tested. We suggest that the increase of degradation rate when adding zeolites in low concentrations is unrelated to the ammonium reduction caused by the zeolite.

  • 19. Nordell, Erik
    et al.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Post digestion of biogas production residues at mid-range mesophilic temperature2011Ingår i: International IWA-Symposium on Anaerobic Digestion of Solid Waste and Energy Crops 2011 Vienna, Austria, August 28th – September 1st, IWA Publishing, 2011Konferensbidrag (Refereegranskat)
    Abstract [en]

    A common way to store digestate from anaerobic digesters is in open air lagoons. The aim of this study was to investigate whether cooling of digestate before transfer to the storage prevents methane production. Furthermore, if methanogenesis is not prevented, to determine the potential maximum methane slip from an open air lagoon, supplied with heat exchanged digestate from a mesophilic co-digestion plant such as Linköping biogas plant. Results indicate that methane production is not terminated by cooling and that a high methane production can occur in open air lagoons if the conditions are advantageous. Furthermore, the results suggest that it can be worthwhile, from both an economical and an environmental point, to replace open air lagoons with closed post-digesting units. At 25 oC, the methane slip from an open air lagoon could reach as high as 2.6% of the total methane production of a biogas plant, even when the volume of the open air lagoon is only one third of the digesters volume. The combination of low additional cost of production, with significant decrease in release of green house gases to the atmosphere, makes the implementation of post-digestion units at larger biogas plants attractive.

  • 20.
    Speda, Jutta
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Johansson, Mikaela
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Odnell, Anna
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten. Karshult Municipal Waste Water Treatment Plant, Sweden.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten. InZymes Biotech AB, Gjuterigatan 1B, S-58273 Linkoping, Sweden.
    Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes2017Ingår i: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 10, artikel-id 129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Enzymatic treatment of lignocellulosic material for increased biogas production has so far focused on pretreatment methods. However, often combinations of enzymes and different physicochemical treatments are necessary to achieve a desired effect. This need for additional energy and chemicals compromises the rationale of using enzymes for low energy treatment to promote biogas production. Therefore, simpler and less energy intensive in situ anaerobic digester treatment with enzymes is desirable. However, investigations in which exogenous enzymes are added to treat the material in situ have shown mixed success, possibly because the enzymes used originated from organisms not evolutionarily adapted to the environment of anaerobic digesters. In this study, to examine the effect of enzymes endogenous to methanogenic microbial communities, cellulolytic enzymes were instead overproduced and collected from a dedicated methanogenic microbial community. By this approach, a solution with very high endogenous microbial cellulolytic activity was produced and tested for the effect on biogas production from lignocellulose by in situ anaerobic digester treatment. Results: Addition of enzymes, endogenous to the environment of a mixed methanogenic microbial community, to the anaerobic digestion of ensiled forage ley resulted in significantly increased rate and yield of biomethane production. The enzyme solution had an instant effect on more readily available cellulosic material. More importantly, the induced enzyme solution also affected the biogas production rate from less accessible cellulosic material in a second slower phase of lignocellulose digestion. Notably, this effect was maintained throughout the experiment to completely digested lignocellulosic substrate. Conclusions: The induced enzyme solution collected from a microbial methanogenic community contained enzymes that were apparently active and stable in the environment of anaerobic digestion. The enzymatic activity had a profound effect on the biogas production rate and yield, comparable with the results of many pretreatment methods. Thus, application of such enzymes could enable efficient low energy in situ anaerobic digester treatment for increased biomethane production from lignocellulosic material.

  • 21.
    Svedhem, Sofia
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Enander, Karin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Sjöbom, Hans
    Biacore AB, Uppsala, Sweden.
    Liedberg, Bo
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Tillämpad Fysik. Linköpings universitet, Tekniska högskolan.
    Löfås, Stefan
    Biacore AB, Uppsala, Sweden.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Sjöstrand, Sven-Erik
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Svensson, Stefan
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Lundström, Ingemar
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Tillämpad Fysik. Linköpings universitet, Tekniska högskolan.
    Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor2001Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 296, nr 2, s. 188-196Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s−1. For the mutants, dissociation rates were faster (0.022–0.025 s−1), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

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