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Biophysical studies of protein folding upon interaction with molecular chaperones
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
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: urn:nbn:se:liu:diva-51604ISBN: 978‐91‐7393‐497‐8 OAI: oai:DiVA.org:liu-51604DiVA: diva2:275972
Public defence
2009-11-27, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2009-11-09 Created: 2009-11-09 Last updated: 2012-11-15Bibliographically approved
List of papers
1. A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding
Open this publication in new window or tab >>A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding
2009 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, no 2, 475-479 p.Article in journal (Refereed) Published
Abstract [en]

The protein folding process is often in vitro rate-limited by slow cis-trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active-site located Arg 55 is not required for catalysis of the rate-limiting prolyl cis-trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis-trans proline isomerization in HCA II occurs in the active-site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding-assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.

Keyword
cis-trans proline isomerization, cyclophilin 18, prolyl isomerases, human carbonic anhydrase II
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-17882 (URN)10.1002/pro.28 (DOI)
Available from: 2009-04-25 Created: 2009-04-24 Last updated: 2017-12-13Bibliographically approved
2. Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates
Open this publication in new window or tab >>Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates
Show others...
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.

Keyword
Chaperone, carbonic anhydrase, citrate synthase, peptidyl‐prolyl cis/trans isomerase, proline isomerase, cyclophilin
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-51602 (URN)10.1021/bi901997q (DOI)20070121 (PubMedID)
Available from: 2009-11-09 Created: 2009-11-09 Last updated: 2017-12-12Bibliographically approved
3. Not merely a passive co-chaperone: dynamic remodeling of protein substrate by GroES alone and in concert with GroEL
Open this publication in new window or tab >>Not merely a passive co-chaperone: dynamic remodeling of protein substrate by GroES alone and in concert with GroEL
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Stopped‐flow folding experiments of human carbonic anhydrase II (HCA II) monitored by ANS fluorescence showed formation of an early molten globule intermediate. Folding of HCA II both in the presence of GroEL alone or GroES alone led to a loss of ANS binding compared to that in the spontaneous refolding process, showing that GroES alone is capable to interact with the refolding protein and that the molten globule substrate seems to be brought into a more unfolded state by both chaperonins. Moreover, an additive effect of the reduction of ANS binding during the early refolding stages was observed in the presence of GroEL+GroES, suggesting a concerted additive decrease in formation of molten globule by the chaperonins. The interactions during folding (from 50 ms to 3 h) between HCA II and GroEL alone, GroES alone, GroEL/ES and GroEL/ES/ATP was monitored in more detail using five fluorescence (AEDANS) labeled HCA II mutants and steady‐state and stopped‐flow Trp‐AEDANS FRET measurements. We observed that GroEL stretches the protein substrate as an early event in the folding process, when compared to spontaneous folding. Interestingly, GroES alone can interact with the folding protein leading to remodelling of the structure of the molten globule intermediate. Furthermore, GroES exerts additive stretching effects of the protein substrate in concert with GroEL. However, in the absence of GroEL the action by GroES is transient and does not affect the reactivation kinetics or final yield and thereby GroES does not exhibit classical chaperone activity, which is likely the reason why the independent GroES activity on protein substrates has gone undiscovered for such a long time.

Keyword
Chaperone, FRET, protein folding, molten‐globule, and carbonic anhydrase
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-51603 (URN)
Available from: 2009-11-09 Created: 2009-11-09 Last updated: 2012-11-15Bibliographically approved
4. Domain-specific chaperone-induced expansion is required for ß-actin folding: a comparison of ß-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC)
Open this publication in new window or tab >>Domain-specific chaperone-induced expansion is required for ß-actin folding: a comparison of ß-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC)
Show others...
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 44, 12639-12647 p.Article in journal (Refereed) Published
Abstract [en]

Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native ß-actin, but is unable to guide ß-actin toward the native state. In this study we identify conformational rearrangements in ß-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of ß-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled ß-actin was characterized, and the structural rearrangement was compared to the case of the ß-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarström, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the ß-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound ß-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the ß-actin molecule. It can be concluded that the specific and directed rearrangement of the ß-actin structure, seen in the natural ß-actin-TRiC system, is vital for guiding ß-actin to the native state. © 2007 American Chemical Society.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-47822 (URN)10.1021/bi700658n (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved

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