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Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
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2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 6, 1137-1145 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2010. Vol. 49, no 6, 1137-1145 p.
Keyword [en]
Chaperone, carbonic anhydrase, citrate synthase, peptidyl‐prolyl cis/trans isomerase, proline isomerase, cyclophilin
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-51602DOI: 10.1021/bi901997qPubMedID: 20070121OAI: oai:DiVA.org:liu-51602DiVA: diva2:275967
Available from: 2009-11-09 Created: 2009-11-09 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Biophysical studies of protein folding upon interaction with molecular chaperones
Open this publication in new window or tab >>Biophysical studies of protein folding upon interaction with molecular chaperones
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are biological macromolecules that serve all functions in cells. Every protein consists of a sequence of amino acids that is folded into a three‐dimensional structure to maintain the unique information it contains and to allow the protein to perform its specific actions. Improper folding caused by mutations in the amino acid sequence or environmental stress can lead to protein aggregation and ultimately to protein conformational disorders such as Parkinson’s disease and other dreadful diseases. Nature has developed special classes of protein guards called foldases and chaperones that can increase folding efficiency in the crowded intracellular milieu by preventing protein aggregation. The present research was aimed to elucidate how chaperones and foldases interact with their target proteins during folding. Special attention was focused on refolding kinetics and dynamic remodulation of site‐specific labeled cysteine variants of the protein human carbonic anhydrase (HCA II) upon interaction with the PPIase cyclophilin18 (Cyp18) and the chaperonin GroEL. Part of the work also compared properties of the group I chaperonin GroEL and the group II chaperonin TRiC, considering how they mediate structural alterations uponinteraction with the cytoskeletal target protein β‐actin. These interactions were studied by various fluorescence techniques, including fluorescence resonance energy transfer (FRET) and fluorescence anisotropy.

Refolding of HCA II is an extremely complicated process that involves very fast and slow folding events, and research has shown that Cyp18 enhances the slow rate‐limiting cistrans proline isomerization steps during the refolding process. Furthermore, the active‐site mutant Cyp18R55A has been reported to posses only about 1% catalytic efficiency when acting on short chromogenic peptide substrates. However, we found that Cyp18R55A is as efficient as the wild‐type Cyp18 in accelerating HCA II refolding. We also noted that Cyp18 enhanced the final yield of the severely destabilized HCA IIH107N, and HCA IIH107F mutants by rescuing transient molten globule intermediates from misfolding as a result of condensation of hydrophobic patches at very early stages of the folding process. These findings led to the conclusion that Arg 55, located in the active site of Cyp18, is not required for prolyl cistrans isomerization of protein substrates, and that Cyp18 can function as both a folding catalyst and a chaperone during HCA II folding.

Studies have demonstrated that sequestering of protein substrates by the chaperonin GroEL alone results in binding‐induced unfolding of aggregation‐prone molten globule intermediates. It was previously assumed that the co‐chaperonin GroES does not play an independent role in folding. However, based on FRET measurements, we found that GroEL alone stretches the protein substrate as an early event, and also that GroES alone can transiently remodulate the structure of the molten globule intermediate during the refolding process. In addition, GroES acts in i concert with GroEL to exert additive transient stretchng effects on the protein core, and it reverses the unfoldase activity of the GroEL termini, leading to compaction of the structure to attain the more constrained native state.

Earlier investigations have shown that partially folded β‐actin binds to both GroEL and the TRiC chaperonin. However, only TRiC guides correct folding of β‐actin, whereas the GroEL–β‐actin interaction is non‐productive. Homo‐FRET measurements on β‐actin mutants labeled with fluorescein during interaction with GroEL and TRiC indicated that interplay with both the chaperonins lead to binding‐induced unfolding and dynamic remodulation of β‐actin. More specifically, the interaction with TRiC resulted in considerable expansion of the entrance of the ATP‐binding cleft of β‐actin by effecting specific modulation of the β‐actin sub‐domains followed by the formation of a compressed state (native‐like) during release from TriC. Conformational rearrangements of β‐actin by GroEL on the other and were ore modest. β‐actin remained rather compact in the complex and consequently did not lead to the native‐like state ven in the encapsulated cis‐cavity when capped by GroES.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 82 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1285
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-51604 (URN)978‐91‐7393‐497‐8 (ISBN)
Public defence
2009-11-27, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
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Available from: 2009-11-09 Created: 2009-11-09 Last updated: 2012-11-15Bibliographically approved

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Moparthi, Satish BabuFristedt, RikardMishra, RajeshAlmstedt, KarinKarlsson, MartinHammarström, PerCarlsson, Uno

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