DOCSLIB.ORG

Characterization of the Dnak-Dnaj-Grpe System Under Oxidative Heat Stress

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterization of the Dnak-Dnaj-Grpe System Under Oxidative Heat Stress Institut für Organische Chemie und Biochemie Lehrstuhl für Biotechnologie Characterization of the DnaK-DnaJ-GrpE system under oxidative heat stress Katrin Linke Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzende: Univ.-Prof. Dr. S. Weinkauf Prüfer der Dissertation: 1. Univ.-Prof. Dr. J. Buchner 2. Asst.-Prof. U. Jakob, Ph.D., University of Michigan, USA Die Dissertation wurde am 17.01.2005 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 17.02.2005 angenommen. i Contents 1 SUMMERY...........................................................................................................1 2 INTRODUCTION..................................................................................................3 2.1 About the Ups and Downs of proteins ........................................................................3 2.1.1 Protein folding in vivo ............................................................................................3 2.1.2 Chaperones – Helpers in hard times.......................................................................4 2.1.3 The many classes of molecular chaperones............................................................5 2.2 Heat shock response and its regulation ......................................................................7 2.3 The DnaK/DnaJ/GrpE-system ....................................................................................8 2.3.1 The molecular chaperone DnaK.............................................................................9 2.3.2 The molecular chaperone DnaJ ............................................................................10 2.3.3 Mechanism of the DnaK/DnaJ/GrpE-system .......................................................13 2.4 Aerobic life and oxidative stress................................................................................14 2.4.1 Redox regulation of protein activity.....................................................................17 2.5 Objective......................................................................................................................21 3 MATERIAL AND METHODS .............................................................................22 3.1 Material .......................................................................................................................22 3.1.1 Strains...................................................................................................................22 3.1.2 Plasmids................................................................................................................22 3.1.3 Primer ...................................................................................................................23 3.1.3.1 Mutagenesis Primer ..........................................................................................23 3.1.3.2 Sequencing Primer............................................................................................23 3.1.4 Proteins.................................................................................................................23 3.1.5 Antibodies, Markers, Dyes, Antibiotics and Inhibitors........................................24 3.1.6 Chemicals .............................................................................................................24 3.1.7 Buffers and Solutions ...........................................................................................26 3.1.8 Kits and Chromatography Material ......................................................................27 3.1.9 Other Material......................................................................................................27 3.1.10 Technical Equipment............................................................................................28 3.1.11 Software................................................................................................................29 3.2 Molecular-biological methods ...................................................................................29 3.2.1 Cultivation and conservation of E. coli strains.....................................................29 3.2.2 QuikChange site-directed mutagenesis ................................................................29 3.2.2.1 Construction of DnaJ zinc center mutants........................................................30 3.2.2.2 Construction of the DnaKCys15Ala-mutant..........................................................31 3.2.3 Preparation and transformation of heat competent cells ......................................31 3.3 Preparative methods ..................................................................................................31 3.3.1 Ammonium sulfate precipitation..........................................................................32 3.3.2 Chromatography ...................................................................................................32 ii 3.3.3 Concentration and dialysis ...................................................................................33 3.3.4 Purification of ATP-depleted DnaK.....................................................................34 3.3.5 Purification of DnaJ..............................................................................................35 3.3.6 Purification of GrpE.............................................................................................37 3.4 Protein-biochemical methods ....................................................................................38 3.4.1 Determination of protein concentration................................................................38 3.4.2 Oxidation and reduction of DnaJ..........................................................................39 3.4.3 Inactivation and reactivation of DnaK in vitro.....................................................40 3.4.3.1 Oxidation of DnaK ...........................................................................................40 3.4.3.2 Reduction of DnaK...........................................................................................40 3.4.4 Determination of free thiol groups in proteins .....................................................40 3.4.4.1 Ellman’s assay..................................................................................................40 3.4.4.2 PAR-PMPS assay.............................................................................................41 3.4.4.3 AMS trapping of DnaJ......................................................................................41 3.4.4.4 Biotinylation of DnaK ......................................................................................42 3.4.5 SDS PAGE and Protein Staining..........................................................................43 3.4.6 Immunoblotting (western blotting).......................................................................44 3.5 Spectroscopic methods ...............................................................................................45 3.5.1 Fluorescence Measurements of DnaK..................................................................45 3.5.2 Circular dichroism measurements ........................................................................45 3.6 Chaperone assays in vitro...........................................................................................46 3.6.1 Light scattering experiments ................................................................................46 3.6.1.1 Aggregation of chemically denatured luciferase..............................................47 3.6.1.2 Aggregation of chemically denatured citrate synthase.....................................47 3.6.2 Reactivation of chemically denatured luciferase..................................................47 3.6.3 Reactivation of chemically denatured citrate synthase.........................................48 3.6.4 Determination of DnaK’s ATPase activity...........................................................48 3.6.4.1 Steady state ATPase assay................................................................................49 3.6.4.2 Single turn over ATPase assay.........................................................................49 3.7 Phenotypic assays and other in vivo methods ..........................................................49 3.7.1 Growth under stress conditions ............................................................................49 3.7.2 Cell motility assay ................................................................................................50 3.7.3 Phage λ replication assay .....................................................................................50 3.7.4 Determination of cellular ATP level ....................................................................50 4 RESULTS ..........................................................................................................52 4.1 Characterization of DnaJ under oxidative stress conditions..................................52 4.1.1 DnaJ – Chaperone with two zinc centers .............................................................52 4.1.2 Zinc content of DnaJ in vitro................................................................................52 4.1.3 Oxidation of DnaJ in vitro....................................................................................53
Load more

Recommended publications
  • Reciprocal Dependence Between the Thioredoxin-Like Activity of Dnaj and the Polypeptide-Unfolding Activity of Dnak
    ORIGINAL RESEARCH ARTICLE published: 31 July 2014 MOLECULAR BIOSCIENCES doi: 10.3389/fmolb.2014.00007 Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK Rayees U. H. Mattoo , America Farina Henriquez Cuendet , Sujatha Subanna , Andrija Finka , Smriti Priya , Sandeep K. Sharma and Pierre Goloubinoff* DBMV, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland Edited by: The role of bacterial Hsp40, DnaJ, is to co-chaperone the binding of misfolded or Rui Joaquim Sousa, University of alternatively folded proteins to bacterial Hsp70, DnaK, which is an ATP-fuelled unfolding Texas Health Science Center, USA chaperone. In addition to its DnaK targeting activity, DnaJ has a weak thiol-reductase Reviewed by: activity. In between the substrate-binding domain and the J-domain anchor to DnaK, Kürsad Turgay, Leibniz Universität 2+ Hannover, Germany DnaJ has a unique domain with four conserved CXXC motives that bind two Zn and Kevin Anthony Morano, UTHealth partly contribute to polypeptide binding. Here, we deleted in DnaJ this Zn-binding domain, Houston, USA which is characteristic to type I but not of type II or III J-proteins. This caused a loss *Correspondence: of the thiol-reductase activity and strongly reduced the ability of DnaJ to mediate the Pierre Goloubinoff, DBMV, Faculty of ATP- and DnaK-dependent unfolding/refolding of mildly oxidized misfolded polypeptides, Biology and Medicine, University of Lausanne, Biophore Building, an inhibition that was alleviated in the presence of thioredoxin or DTT. We suggest 1015-Lausanne, Switzerland that in addition to their general ability to target misfolded polypeptide substrates to e-mail: [email protected] the Hsp70/Hsp110 chaperone machinery, Type I J-proteins carry an ancillary protein dithiol-isomerase function that can synergize the unfolding action of the chaperone, in the particular case of substrates that are further stabilized by non-native disulfide bonds.
    [Show full text]
  • Groel Actively Stimulates Folding of the Endogenous Substrate Protein Pepq
    ARTICLE Received 2 Oct 2016 | Accepted 13 May 2017 | Published 30 Jun 2017 DOI: 10.1038/ncomms15934 OPEN GroEL actively stimulates folding of the endogenous substrate protein PepQ Jeremy Weaver1,*,w, Mengqiu Jiang1,2,*, Andrew Roth1, Jason Puchalla3, Junjie Zhang1 & Hays S. Rye1 Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role. 1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77845, USA. 2 State Key Laboratory of Biocontrol, School of Life Science, Sun Yat-sen University, Guangzhou, Guangdong 510275, China. 3 Department of Physics, Princeton University, Princeton, New Jersey 08544, USA. * These authors contributed equally to this work. w Present address: Division of Molecular and Cellular Biology, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
    [Show full text]
  • The Holdase Function of Escherichia Coli Hsp70 (Dnak) Chaperone
    bioRxiv preprint doi: https://doi.org/10.1101/305854; this version posted April 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The holdase function of Escherichia coli Hsp70 (DnaK) chaperone Ricksen S. Winardhi,1, 2, ∗ Qingnan Tang,1, 2, ∗ Huijuan You,2 Michael Sheetz,2 and Jie Yan1, 2, 3, y 1Department of Physics, National University of Singapore, Singapore 117542 2Mechanobiology Institute, National University of Singapore, Singapore 117411 3Centre for Bioimaging Sciences, National University of Singapore, Singapore 117546 (Dated: April 21, 2018) In Escherichia coli, the DnaK/DnaJ/GrpE system plays a critical role in mediating protein re- folding and buffering against protein aggregation due to environmental stress. The underlying mechanism remains unclear. In this work, we probe the activity of DnaK/DnaJ/GrpE system with single-molecule protein refolding assay using tandem repeats of titin immunoglobulin 27 (I27)8. We provide direct evidence that DnaK in apo- and ADP-bound state is predominantly a holdase, which kinetically stabilizes the polyprotein in its unfolded form. Binding of ATP relieves DnaK's holding, allowing protein refolding. The presence of co-chaperone DnaJ and GrpE modulates this holding- release switching, possibly by altering DnaK's nucleotide state. Our findings thus provide important insights to the molecular mechanism of DnaK/DnaJ/GrpE system. INTRODUCTION Hsp70 is of special interest due to its implication in var- ious neurodegenerative diseases, as many of these are In virtually all living organisms, proteins are synthe- caused by accumulation of aggregates/misfolded proteins sized by a molecular machine called ribosome by sequen- [3].
    [Show full text]
  • Redox Response of Iron-Sulfur Glutaredoxin GRXS17 Activates Its Holdase Activity To
    bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896506; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Redox response of iron-sulfur glutaredoxin GRXS17 activates its holdase activity to protect plants from heat stress Laura Martins1,2,#, Johannes Knuesting3,#, Laetitia Bariat1,2, Avilien Dard1,2, Sven A. Freibert4, 5 Christophe H. Marchand5, David Young6,7,8, Nguyen Ho Thuy Dung6,7,8, Anne Debures1,2, Julio Saez-Vasquez1,2, Stéphane D. Lemaire5, Roland Lill4, Joris Messens6,7,8, Renate Scheibe3, Jean- Philippe Reichheld1,2,*, Christophe Riondet1,2 1 Laboratoire Génome et Développement des Plantes, Université Perpignan Via Domitia, F-66860 10 Perpignan, France; 2 Laboratoire Génome et Développement des Plantes, CNRS, F-66860 Perpignan, France; 3 Department of Plant Physiology, FB5, University of Osnabrück, D–49069 Osnabrueck, Germany; 4 Institut für Zytobiologie und Zytopathologie, Philipps-Universität, Robert-Koch-Strasse 6, Marburg 35032, Germany; 15 5 Institut de Biologie Physico-Chimique, UMR8226, CNRS, Sorbonne Université, F-75005 Paris, France; 6 VIB-VUB Center for Structural Biology, 1050 Brussels, Belgium; 7 Brussels Center for Redox Biology, 1050 Brussels, Belgium; 8 Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; 20 # These authors contributed equally to this work. * To whom correspondence should be addressed: E-mail: [email protected]. Jean-Philippe Reichheld, Tel: +33 4 68662225; Fax: +33 4 68668499; E-mail: [email protected]. Laboratoire Génome et Développement des Plantes, Université Perpignan Via Domitia, F-66860 25 Perpignan, France.
    [Show full text]
  • Identification of a Redoxregulated Chaperone Network
    The EMBO Journal (2004) 23, 160–168 | & 2004 European Molecular Biology Organization | All Rights Reserved 0261-4189/04 www.embojournal.org THE EMBO JOJOURNALUR NAL Identification of a redox-regulated chaperone network 1 1 Jo¨ rg H Hoffmann , Katrin Linke , Paul CF served cysteine residues in the C-terminal part of Hsp33. Graf 2, Hauke Lilie3 and Ursula Jakob1,* Under normal reducing conditions, these four cysteines co- ordinate one zinc ion (Jakob et al, 2000). In this state, Hsp33 1Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA, 2Program in Cellular and is monomeric and the chaperone function is downregulated. Molecular Biology, University of Michigan, MI, USA and 3Department of Upon exposure of Hsp33 to oxidizing conditions such as Biotechnology, University of Halle, Halle, Germany H2O2, two intramolecular disulfide bonds are rapidly formed (Barbirz et al, 2000). This appears to cause major conforma- We have identified and reconstituted a multicomponent tional rearrangements in the protein and triggers the dimer- redox-chaperone network that appears to be designed to ization of two oxidized Hsp33 molecules (Graumann et al, protect proteins against stress-induced unfolding and to 2001). Oxidized Hsp33 dimers recognize and interact with refold proteins when conditions return to normal. The folding intermediates of other proteins and very efficiently central player is Hsp33, a redox-regulated molecular cha- prevent their irreversible aggregation processes in vitro. In perone. Hsp33, which is activated by disulfide bond for- vivo, Hsp33 has been shown to play an important role in the mation and subsequent dimerization, works as an efficient defense of bacteria against oxidative stress (Jakob et al, chaperone holdase that binds to unfolding protein inter- 1999).
    [Show full text]
  • Mechanism of the Small ATP-Independent Chaperone Spy Is Substrate Specific
    ARTICLE https://doi.org/10.1038/s41467-021-21120-8 OPEN Mechanism of the small ATP-independent chaperone Spy is substrate specific Rishav Mitra1,2, Varun V. Gadkari3, Ben A. Meinen1,2, Carlo P. M. van Mierlo 4, Brandon T. Ruotolo 3 & ✉ James C. A. Bardwell1,2 ATP-independent chaperones are usually considered to be holdases that rapidly bind to non- native states of substrate proteins and prevent their aggregation. These chaperones are 1234567890():,; thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy’s action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action. 1 Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA. 2 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. 3 Department of Chemistry, University of Michigan, Ann Arbor, MI, USA. 4 Laboratory of Biochemistry, Wageningen ✉ University, Wageningen, The Netherlands. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:851 | https://doi.org/10.1038/s41467-021-21120-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21120-8 opologically complex proteins often populate misfolded bound to the chaperone10.
    [Show full text]
  • Intrinsic Unfoldase/Foldase Activity of the Chaperonin Groel Directly Demonstrated Using Multinuclear Relaxation-Based
    Intrinsic unfoldase/foldase activity of the chaperonin INAUGURAL ARTICLE GroEL directly demonstrated using multinuclear relaxation-based NMR David S. Libich1, Vitali Tugarinov1, and G. Marius Clore2 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014. Contributed by G. Marius Clore, May 27, 2015 (sent for review May 13, 2015) The prototypical chaperonin GroEL assists protein folding through than 1%. Further, GroEL unfoldase/foldase activity is modu- an ATP-dependent encapsulation mechanism. The details of how lated by SH3 deuteration, indicating that catalysis of the exchange GroEL folds proteins remain elusive, particularly because encapsu- reaction between folded and intermediate states involves di- lation is not an absolute requirement for successful re/folding. rect interaction of the substrate with the walls of the GroEL Here we make use of a metastable model protein substrate, com- chambers. These results provide a basis for how chaperonins, prising a triple mutant of Fyn SH3, to directly demonstrate, by in the absence of cofactors and encapsulation, may be able to simultaneous analysis of three complementary NMR-based relax- passively protect the cell from the deleterious effects of misfolded ation experiments (lifetime line broadening, dark state exchange protein accumulation. saturation transfer, and Carr–Purcell–Meinboom–Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion Results and Discussion rate between the native state and a well-defined folding interme- 15N-Relaxation–Based NMR Measurements.
    [Show full text]
  • Groel-Assisted Protein Folding: Groes Binding-Induced Displacement of Denatured Proteins from Groel to Bulk Solution
    biomolecules Article Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution Victor Marchenkov 1, Andrey Gorokhovatsky 2, Natalia Marchenko 1, Tanya Ivashina 3 and Gennady Semisotnov 1,* 1 Institute of Protein Research, Russian Academy of Sciences, Institutskaya Street 4, 142290 Pushchino, Russia; [email protected] (V.M.); [email protected] (N.M.) 2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ulitsa Miklukho-Maklaya 16/10, GSP-7, 117997 Moscow, Russia; [email protected] 3 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, 142290 Pushchino, Russia; [email protected] * Correspondence: [email protected]; Tel.: +7-495-514-0218 Received: 29 December 2019; Accepted: 18 January 2020; Published: 20 January 2020 Abstract: The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed.
    [Show full text]
  • Folding While Bound to Chaperones
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Curr Opin Manuscript Author Struct Biol. Author Manuscript Author manuscript; available in PMC 2019 February 01. Published in final edited form as: Curr Opin Struct Biol. 2018 February ; 48: 1–5. doi:10.1016/j.sbi.2017.06.009. Folding while bound to chaperones Scott Horowitz1, Philipp Koldewey2, Frederick Stull2,3, and James CA Bardwell2,3 1Department of Chemistry & Biochemistry and the Knoebel Institute for Healthy Aging, University of Denver, 2155 E. Wesley Avenue, Denver, CO 80208, USA 2Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA 3Howard Hughes Medical Institute, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA Abstract Chaperones are important in preventing protein aggregation and aiding protein folding. How chaperones aid protein folding remains a key question in understanding their mechanism. The possibility of proteins folding while bound to chaperones was reintroduced recently with the chaperone Spy, many years after the phenomenon was first reported with the chaperones GroEL and SecB. In this review, we discuss the salient features of folding while bound in the cases for which it has been observed and speculate about its biological importance and possible occurrence in other chaperones. Introduction Molecular chaperones are responsible for keeping proteins in their folded form and preventing protein aggregation in the cell [1]. Defects in protein folding can lead to myriad diseases, including Alzheimer’s [2], Parkinson’s [3], and Huntington's disease [4]. Although much has been learned in recent years, our current understanding of how chaperones aid protein folding remains incomplete.
    [Show full text]
  • Intrinsic Unfoldase/Foldase Activity of the Chaperonin Groel Directly
    Intrinsic unfoldase/foldase activity of the chaperonin INAUGURAL ARTICLE GroEL directly demonstrated using multinuclear relaxation-based NMR David S. Libich1, Vitali Tugarinov1, and G. Marius Clore2 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014. Contributed by G. Marius Clore, May 27, 2015 (sent for review May 13, 2015) The prototypical chaperonin GroEL assists protein folding through than 1%. Further, GroEL unfoldase/foldase activity is modu- an ATP-dependent encapsulation mechanism. The details of how lated by SH3 deuteration, indicating that catalysis of the exchange GroEL folds proteins remain elusive, particularly because encapsu- reaction between folded and intermediate states involves di- lation is not an absolute requirement for successful re/folding. rect interaction of the substrate with the walls of the GroEL Here we make use of a metastable model protein substrate, com- chambers. These results provide a basis for how chaperonins, prising a triple mutant of Fyn SH3, to directly demonstrate, by in the absence of cofactors and encapsulation, may be able to simultaneous analysis of three complementary NMR-based relax- passively protect the cell from the deleterious effects of misfolded ation experiments (lifetime line broadening, dark state exchange protein accumulation. saturation transfer, and Carr–Purcell–Meinboom–Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion Results and Discussion rate between the native state and a well-defined folding interme- 15N-Relaxation–Based NMR Measurements.
    [Show full text]
  • Measuring Protein Stability in the Groel Chaperonin Cage Reveals Massive Destabilization Ilia Korobko1, Hisham Mazal2, Gilad Haran2, Amnon Horovitz1*
    RESEARCH ARTICLE Measuring protein stability in the GroEL chaperonin cage reveals massive destabilization Ilia Korobko1, Hisham Mazal2, Gilad Haran2, Amnon Horovitz1* 1Departments of Structural Biology, Weizmann Institute of Science, Rehovot, Israel; 2Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, Israel Abstract The thermodynamics of protein folding in bulk solution have been thoroughly investigated for decades. By contrast, measurements of protein substrate stability inside the GroEL/ES chaperonin cage have not been reported. Such measurements require stable encapsulation, that is no escape of the substrate into bulk solution during experiments, and a way to perturb protein stability without affecting the chaperonin system itself. Here, by establishing such conditions, we show that protein stability in the chaperonin cage is reduced dramatically by more than 5 kcal molÀ1 compared to that in bulk solution. Given that steric confinement alone is stabilizing, our results indicate that hydrophobic and/or electrostatic effects in the cavity are strongly destabilizing. Our findings are consistent with the iterative annealing mechanism of action proposed for the chaperonin GroEL. Introduction The Escherichia coli GroE chaperonin system, which comprises GroEL and its co-factor GroES, assists protein folding in vivo and in vitro in an ATP-dependent manner (Thirumalai and Lorimer, 2001; Saibil et al., 2013; Hayer-Hartl et al., 2016; Gruber and Horovitz, 2016). Binding of GroES to GroEL forms a cage in which encapsulated substrate proteins can fold in isolation from bulk solution. *For correspondence: The GroE system has been studied intensively for more than three decades, but it is still unclear and [email protected] controversial whether its cavity is a ‘passive cage’ in which protein substrate aggregation is pre- vented but the folding pathway is unchanged or a chamber in which the folding process is altered in Competing interests: The some manner.
    [Show full text]
  • Enzymes As Chaperones and Chaperones As Enzymes
    FEBS Letters 425 (1998) 382^384 FEBS 20039 Minireview Enzymes as chaperones and chaperones as enzymes Chih-Chen Wang, Chen-Lu Tsou* National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing 100101, People's Republic of China Received 9 February 1998 for a protein to be a molecular chaperone. In addition, for Abstract Chaperones and foldases are two groups of accessory proteins which assist maturation of nascent peptides into PDI to promote the joining of thiol groups distantly situated functional proteins in cells. Protein disulfide isomerase, a in the peptide sequence to form correct disul¢des the peptide foldase, and ATP-dependent proteases, responsible for degrada- chain has to be folded at least to some extent to bring the tion of misfolded proteins in cells, both have intrinsic chaperone thiol groups together. The spontaneous folding of the peptide activities. Trigger factor and DnaJ, well known Escherichia coli chain and the oxidation of thiol groups in vitro are often slow chaperones, show peptidyl prolyl isomerase and protein disulfide processes and the PDI-catalyzed folding does not need the isomerase activities respectively. It is suggested that the presence of other chaperones. We therefore put forward the combination of chaperone and enzyme activities in one molecule hypothesis that PDI is both an enzyme and a molecular chap- is the result of evolution to increase molecular efficiency. erone [5]. Many in vitro and in vivo data have now accumu- z 1998 Federation of European Biochemical Societies. lated to support the hypothesis that in addition to its isomer- Key words: Foldase; Chaperone; Protein disul¢de isomerase; ase activity in the catalysis of native disul¢de formation PDI Trigger factor; ATP-dependent protease; Protein folding does have intrinsic activity as a chaperone [6^13].
    [Show full text]