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Unfolding a folding...
Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
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- Almstedt, Karin, 1980- (author)
- Linköpings universitet,Biokemi,Tekniska högskolan
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- Lundqvist, Martin (author)
- Linköpings universitet,Molekylär Bioteknik,Tekniska högskolan
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- Carlsson, Jonas (author)
- Linköpings universitet,Bioinformatik,Tekniska högskolan
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- Karlsson, Martin (author)
- Linköpings universitet,Biokemi,Tekniska högskolan
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- Persson, Bengt (author)
- Linköpings universitet,Bioinformatik,Tekniska högskolan
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- Jonsson, Bengt-Harald (author)
- Linköpings universitet,Molekylär Bioteknik,Tekniska högskolan
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- Carlsson, Uno (author)
- Linköpings universitet,Biokemi,Tekniska högskolan
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- Hammarström, Per (author)
- Linköpings universitet,Biokemi,Tekniska högskolan
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(creator_code:org_t)
- Oxford : Elsevier, 2004
- 2004
- English.
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In: Journal of Molecular Biology. - Oxford : Elsevier. - 0022-2836 .- 1089-8638. ; 342:2, s. 619-633
- Related links:
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http://urn.kb.se/res...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Subject headings
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- 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.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Keyword
- Misfolding
- loss-of-function
- aggregation
- molten globule
- misfolding inhibitor
- Biochemistry
- Biokemi
Publication and Content Type
- ref (subject category)
- art (subject category)
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