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Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology. (Per Hammarström)
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-7642-9263
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
2008 (English)In: Biochemistry, ISSN 0006-2960, Vol. 47, no 5, 1288-1298 p.Article in journal (Refereed) Published
Abstract [en]

Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order:  wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion:  (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

Place, publisher, year, edition, pages
Washington: ACS , 2008. Vol. 47, no 5, 1288-1298 p.
Keyword [en]
mutant mapping, carbonic anhydrase, misfolding, loss-of-function, molten globule
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-21056DOI: 10.1021/bi701720pOAI: oai:DiVA.org:liu-21056DiVA: diva2:240487
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2013-10-04Bibliographically approved
In thesis
1. Protein Misfolding in Human Diseases
Open this publication in new window or tab >>Protein Misfolding in Human Diseases
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories:

  1. Loss-of-function diseases
  2. Gain-of-toxic-function diseases
  3. Infectious misfolding diseases

 

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.

 

The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease.

Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 103 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1239
Keyword
Misfolding, carbonic anhydrase, prion protein, protein stability
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-21077 (URN)978-91-7393-698-9 (ISBN)
Public defence
2009-02-26, Planck, Campus Valla, Linköpings universitet, Linköping, 13:15 (English)
Opponent
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Available from: 2009-10-16 Created: 2009-09-28 Last updated: 2009-10-16Bibliographically approved

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Almstedt, KarinMårtensson, Lars-GöranCarlsson, UnoHammarström, Per

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