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  • 1.
    Babu Moparthi, Satish
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Institut Fresnel, CNRS UMR 7249, Aix-Marseille Université, Marseille, France.
    Sjölander, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Villebeck, Laila
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL2014In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 7, no 1, p. 1-15Article, review/survey (Refereed)
    Abstract [en]

    The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.

  • 2.
    Moparhti, Satish Babu
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Arbman, Gunnar
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of surgery. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Wallin, Åsa
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Oncology.
    Kayed, Hany
    General Surgery, University of Heidelberg, Heidelberg, Germany.
    Kleeff, Jörg
    General Surgery, University of Heidelberg, Heidelberg, Germany.
    Zentgraf, Hanswalter
    Applied Tumor Virology, University of Heidelberg, Heidelberg, Germany.
    Sun, Xiao-Feng
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Oncology. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL.
    Expression of MAC30 protein is related to survival and biological variables in primary and metastatic colorectal cancers2007In: International Journal of Oncology, ISSN 1019-6439, Vol. 30, no 1, p. 91-95Article in journal (Refereed)
    Abstract [en]

    MAC30 is highly expressed in several types of tumors including colorectal cancers, however, its clinicopathological and biological significance in colorectal cancers is currently not known. The aim of our study was to investigate MAC30 expression in distant normal mucosa, adjacent normal mucosa, primary tumors and metastases of colorectal cancer, and to determine the relationship between MAC30 expression and clinicopathological and biological variables. MAC30 expression was immunohistochemically examined in distant normal mucosa (n = 54), adjacent normal mucosa (n = 123), primary tumors (n = 217) and lymph node metastases (n = 56) from colorectal cancer patients. MAC30 cytoplasmic expression was increased from distant normal mucosa to primary tumor and to metastasis (p < 0.0001-0.04). Furthermore, 40% primary and 37% metastatic tumors showed stronger cytoplasmic expression of MAC30 at the tumor invasive margins compared to inner tumor areas. Strong cytoplasmic expression of MAC30 in the metastasis was related to a poor prognosis (p = 0.04). MAC30 cytoplasmic expression was positively related to expression of proliferating cell nuclear antigen (p = 0.04), p53 (p = 0.04), nucleoporin 88 (p = 0.001), legumain (p = 0.004) and particularly interesting new cysteine-histidine rich protein (p = 0.004). However, MAC30 expression in the nucleus and stroma did not have any clinicopathological and biological significance (p > 0.05). In conclusion, MAC30 protein may play a role in development of colorectal cancer, and can be considered as a prognostic factor.

  • 3.
    Moparthi, Satish Babu
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Biophysical studies of protein folding upon interaction with molecular chaperones2009Doctoral 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.

    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, p. 475-479Article 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.

    Keywords
    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: 2018-04-25Bibliographically 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, p. 1137-1145Article 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.

    Keywords
    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: 2018-04-25Bibliographically 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.

    Keywords
    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: 2018-04-25Bibliographically 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, p. 12639-12647Article 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: 2018-04-25Bibliographically approved
  • 4.
    Moparthi, Satish Babu
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Bergman, Viveka
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Adell, Gunnar
    Karolinska University Hospital.
    Thorstensson, Sten
    Kalmar Hospital.
    Sun, Xiao-Feng
    Linköping University, Department of Clinical and Experimental Medicine, Oncology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL.
    pRb2/p130 protein in relation to clinicopathological and biological variables in rectal cancers with a clinical trial of preoperative radiotherapy2009In: INTERNATIONAL JOURNAL OF COLORECTAL DISEASE, ISSN 0179-1958, Vol. 24, no 11, p. 1303-1310Article in journal (Refereed)
    Abstract [en]

    pRb2/p130 plays a key role in cell proliferation and is a considerable progress about expression patterns of pRb2/p130 in number of malignancies. However, pRb2/p130 expression and its significance in rectal cancer remain unknown. The purpose of the present study was to investigate pRb2/p130 protein patterns and their correlations with clinicopathological and biological factors in rectal cancer patients with or without preoperative radiotherapy (RT). pRb2/p130 protein was examined by immunohistochemistry in 130 primary tumors, along with the corresponding 61 distant normal mucosa specimens, 85 adjacent normal mucosa specimens, 34 lymph node metastases, and 93 primary tumor biopsies from rectal cancer patients who participated in a Swedish clinical trial of preoperative RT. The pRb2/p130 protein was mainly localized in the cytoplasm of tumor cells. In nonradiated cases, the lack of pRb2/p130 was related to advanced tumor-node-metastases stage, poorer differentiation, weak fibrosis, less inflammatory infiltration, higher Ki-67, and positive Cox-2 expression (p andlt; 0.05). In radiated cases, the lack of pRb2/p130 was related to nonstaining of Cox-2 and survivin (p andlt; 0.05). pRb2/p130 protein in primary tumors tended to be increased after RT (27% vs 16%, p = 0.07). pRb2/p130 was mainly localized in the cytoplasm rather than in the nucleus in rectal cancer. After RT, pRb2/p130 protein seems to be increased in primary tumors, and further the relationship of the pRb2/p130 with the clinicopathological and biological variables changed compared to the nonradiated cases. However, we did not find that the pRb2/p130 was directly related to RT, tumor recurrence, and patients survival.

  • 5.
    Moparthi, Satish Babu
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Fristedt, Rikard
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mishra, Rajesh
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Almstedt, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 6, p. 1137-1145Article in journal (Refereed)
    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.

  • 6.
    Moparthi, Satish Babu
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding2009In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, no 2, p. 475-479Article in journal (Refereed)
    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.

  • 7.
    Moparthi, Satish Babu
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Not merely a passive co-chaperone: dynamic remodeling of protein substrate by GroES alone and in concert with GroELManuscript (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.

  • 8.
    Villebeck, Laila
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Klang, Hanna
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Moparthi, Satish Babu
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lindgren, Mikael
    Department of Physics, The Norwegian University of Science and Technology, 7491 Trondheim, Norway.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Mapping the Different Interactions between Eukaryotic β-actin and the Group I (GroEL) and Group II (TRiC) ChaperoninsManuscript (preprint) (Other academic)
    Abstract [en]

    Productive folding to the native state of the abundant eukaryotic protein actin is dependent on the chaperonin TRiC. The prokaryotic chaperonin GroEL also recognizes actin, but this interaction does not lead to the correct folding of actin. It is well established that GroEL interacts with non-native proteins through hydrophobic interactions. The characteristics of the interactions between TRiC and its target proteins are however unclear. In this study, we present multiple site-directed cysteine labeling and fluorescence measurements indicating that actin initially binds to TRiC through several interaction sites and that the surfaces of the interaction areas on the walls of the TRiC chamber present both polar and hydrophobic residues. At a later stage in the chaperonin cycle, the binding of ATP causes conformational changes in the chaperonin, which leads to a presentation of a more hydrophobic milieu in TRiC chamber. The conformational changes of the chaperonin causes rearrangements of the actin molecule and new interactions are proposed to be formed. Additionally, we show that the initial binding of actin to TRiC leads to a re-modeling of the nucleotide-binding cleft in actin. We also present data indicating that GroEL presents less specific interaction areas towards the bound actin than TRiC does. The interactions between actin and GroEL are tight and of hydrophobic character. No re-modeling of the nucleotide-binding cleft was obtained in the actin-GroEL complex. We conclude that the interactions between actin and TRiC are of both polar and hydrophobic character, the nature of the interactions are different in the prokaryotic and eukaryotic chaperonins, and the rearrangements of the nucleotide binding cleft of actin seen in the chaperonin cycle of TRiC do not occur in GroEL. We suggest that the rearrangements of the nucleotide-binding site in actin are critical for productive folding of actin.

  • 9.
    Villebeck, Laila
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Moparthi, Satish Babu
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Interactions Between the Bacterial β-actin Homologue MreB and the Group I Chaperonin GroEL and Group II Chaperonin TRiCManuscript (preprint) (Other academic)
    Abstract [en]

    This pilot study on the interaction between MreB andthe chaperonins TRiC and GroEL indicates that thefolding of the actin ancestor was facilitated by thechaperonins. From an evolutionary point of view, it isinteresting to investigate the nature of the bindinginteraction between the prokaryotic system MreB-GroELand compare it to the binding interaction between actinand TRiC and the following questions will be addressed:Does MreB refold in a spontaneous manner or is itsfolding dependent on the action of a chaperonin (GroEL)as in the case of actin folding (TRiC)? Does MreB bind ina similar stretched manner to GroEL as actin binds toTRiC (4, 11), or is the “general” binding inducedunfolding sufficient for guiding MreB to the native state?How does the MreB molecule interact with TRiC, is therea similar stretching as for actin? Are there any analoguessequences between actin and TRiC that are recognized byTRiC and/or GroEL?

    Two single variants where cysteines have beenintroduced at positions 69 and 245 in E. coli MreB(Figure 1 B). These positions are situated at the tips of thecorresponding subdomains 2 and 4 of the actin molecule(4, 12). The double variant N69C/V245C has also beenconstructed. The three variants will be produced andlabeled with fluorescein and subsequent homo-FRETmeasurements will be performed on MreB bound toGroEL, TRiC and GroES. The results will be comparedto the results on actin bound to the chaperonins toinvestigate how the chaperonin-dependent folding ofactin homologues has evolved.

  • 10.
    Villebeck, Laila
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Moparthi, Satish Babu
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lindgren, Mikael
    Department of Physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    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)2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 44, p. 12639-12647Article in journal (Refereed)
    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.

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