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The Escherichia Coli Groel Interaction Proteome: Identification and Classification

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The Escherichia Coli Groel Interaction Proteome: Identification and Classification Max-Planck-Institut für Biochemie, Martinsried Abteilung Zelluläre Biochemie The Escherichia coli GroEL Interaction Proteome: Identification and Classification Michael Johannes Kerner 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. Sevil Weinkauf Prüfer der Dissertation: 1. Hon.-Prof. Dr. Wolfgang Baumeister 2. Univ.-Prof. Dr. Johannes Buchner 3. Hon.-Prof. Dr. Franz-Ulrich Hartl Ludwig-Maximilians-Universität München Die Dissertation wurde am 04.07.2005 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 15.11.2005 angenommen. Danksagung Diese Arbeit wurde in der Zeit von Dezember 1999 bis Dezember 2004 in der Abteilung Zelluläre Biochemie des Max-Planck-Instituts für Biochemie in Martinsried angefertigt. Mein besonderer Dank gilt Prof. Dr. F. Ulrich Hartl für seine hervorragende Betreuung und stete Unterstützung, die Bereitstellung des spannenden Themas und die ausgezeichneten Arbeitsbedingungen in seiner Abteilung. Weiterhin möchte ich Dr. Manajit Hayer-Hartl für zahlreiche hilfreiche Diskussionen und Ratschläge danken. Prof. Dr. Wolfgang Baumeister danke ich herzlich für seine Bereitschaft zur Vertretung dieser Dissertation vor der Technischen Universität München. Ganz besonders danken möchte ich Dr. Dean Naylor für seine ausgezeichnete Betreuung und hervorragende Zusammenarbeit, ohne die diese Arbeit so nicht möglich gewesen wäre. Ebenfalls sehr herzlich danken möchte ich Tobias Maier für die erfolgreiche und sehr gute Zusammenarbeit an diesem Projekt. Beide sind mir nicht nur gute Kollegen, sondern auch sehr gute Freunde. Auch Dr. Anna Stines und Hung-Chun Chang bin ich sehr dankbar für die fruchtbare Zusammenarbeit im Rahmen des vorliegenden Projektes. Dr. Yasushi Ishihama, Prof. Dr. Matthias Mann, Morten Kirkegaard (Syddansk Univer- sitet, Odense, Dänemark) und Prof. Dr. Dmitrij Frishman (Technische Universität Mün- chen und GSF Neuherberg) bin ich sehr dankbar für die äußerst erfolgreiche Kooperation an diesem Projekt und ihre stete Bereitschaft zu weiteren zusätzlichen Analysen. Ebenfalls bedanken möchte ich mich bei Prof. Dr. Costa Georgopoulos (Université de Ge- nève), Dr. Luis Serrano (EMBL Heidelberg), Prof. Dr. Andrei Lupas und Dr. Johannes Soeding (MPI für Entwicklungsbiologie, Tübingen) für ihre Hilfe und Unterstützung wäh- rend dieser Arbeit. Herzlich danken möchte ich auch besonders Dr. José Manuel Barral für seine Unterstüt- zung, seine zahlreichen Hilfestellungen und guten Ratschläge und außerdem für unsere sehr gute Freundschaft. Weiterhin danke ich allen Mitarbeitern der Abteilung Zelluläre Bio- chemie für ihre Hilfe und die gute Atmosphäre. Ich habe hier viele gute Freunde gewonnen. Von ganzem Herzen danke ich auch meiner Familie. Meine Eltern Rotraud und Wolfgang Kerner und meine Geschwister Daniela und Andrea waren mir immer eine große Unter- stützung. Ganz besonders herzlich danke ich meiner Frau Sabine Kerner, die mir mit ihrer Hilfe und Liebe und ihrem großen Verständnis immer zur Seite steht und ohne die mir diese Arbeit sicher um ein Vielfaches schwerer gefallen wäre. i ii Contents 1 Summary 1 2 Introduction 3 2.1 Protein folding 3 2.1.1 Protein structure 3 2.1.2 The protein folding problem 3 2.1.3 Protein folding mechanisms 5 2.1.4 Folding energy landscapes 6 2.2 Protein folding in the cell 6 2.2.1 Protein aggregation and the excluded volume effect 7 2.2.2 De novo protein folding: necessity of molecular chaperones 9 2.3 Molecular chaperone systems for de novo protein folding 10 2.3.1 Overview of the substrate flux through chaperone networks in the cytosol 10 2.3.2 Ribosome-associated chaperones 10 2.3.3 The Hsp70 system 12 2.3.4 The chaperonins 13 2.4 The E. coli chaperonin GroEL/GroES 16 2.4.1 Structure and function 16 2.4.2 Mechanisms of GroEL-mediated protein folding 19 2.4.3 Substrates of GroEL 21 2.5 Aim of the study 23 3 Materials and Methods 24 3.1 Materials 24 3.1.1 Chemicals 24 3.1.2 Instruments 24 3.1.3 Media and buffers 25 3.1.4 Bacterial strains and plasmids 26 3.1.5 Proteins 27 3.2 Molecular biological methods 27 3.2.1 Preparation and transformation of competent E. coli cells 27 3.2.2 DNA analytical methods 28 3.2.3 PCR amplification 28 3.2.4 DNA restriction and ligation 28 3.2.5 Plasmid purification 29 3.3 Protein biochemical methods 29 3.3.1 Protein expression and purification 29 3.3.2 Protein analytical methods 31 3.4 Biochemical and biophysical methods 33 3.4.1 Protein refolding and activity assays 33 3.4.2 In vivo co-expression of chaperones with substrates 34 iii 3.4.3 GroEL/GroES depletion 35 3.4.4 In vivo capture of GroEL/GroES cis-cavity substrates 35 3.4.5 Proteinase K digestion of GroEL/GroES/substrate complexes 36 3.4.6 Sample preparation for protein identification by mass spectrometry 36 3.4.7 Coupled liquid chromatography – mass spectrometry system (LC-MS/MS) 37 3.4.8 Mass spectrometry data analysis 37 3.5 Bioinformatic methods 38 3.5.1 Structural comparison of GroE substrates 38 3.5.2 Protein sequence analyses 38 4 Results 40 4.1 Classification of GroEL dependence by in vitro refolding assays 40 4.1.1 Class I proteins are able to fold spontaneously 40 4.1.2 Class II proteins utilize DnaK or GroEL for folding 43 4.1.3 Class III proteins can be stabilized by DnaK but fold only upon transfer to GroEL 45 4.2 The dependence of substrates on GroEL in vivo 48 4.2.1 Overexpression of GroEL substrates in E. coli 48 4.2.2 Cellular depletion of GroEL/GroES results in misfolding of obligate substrates 49 4.3 Identification of the in vivo GroEL/GroES substrate proteome 51 4.3.1 GroEL/GroES complexes contain encapsulated and trans ring-bound substrates 54 4.3.2 GroEL interacting proteins are enriched in class III proteins 55 4.3.3 General properties of GroEL interacting proteins 58 4.3.4 Class III GroEL substrates are enriched in the TIM (βα)8 barrel fold 61 4.3.5 Functional categories among GroEL interacting proteins 67 4.3.6 Concentration of GroEL substrate proteins in the E. coli lysate and in GroEL/GroES/substrate complexes 68 5 Discussion 73 5.1 Classification of GroEL dependence 73 5.1.1 Folding of class III GroEL substrates 75 5.1.2 Substrates too large for encapsulation inside the GroEL/ GroES cavity 76 5.2 The GroEL interactome 77 5.2.1 Completeness and quality of the dataset 77 5.2.2 Overlap with previously identified GroEL interactors 77 5.2.3 Substrate classification of the GroEL interaction proteome 78 5.2.4 Class III substrate enrichment on GroEL 78 5.2.5 Interaction of trigger factor and DnaK with GroEL 79 5.2.6 The essential role of GroEL 80 5.2.7 GroEL substrate solubility in S. cerevisiae 80 iv 5.3 Properties of GroEL interacting proteins 81 5.3.1 Size dependence 81 5.3.2 Aggregation propensity 82 5.3.3 Structures of GroEL substrates: the TIM (βα) barrel 82 5.3.4 Additional structures of GroEL substrates 84 5.4 GroEL substrate homologs in GroEL-deficient organisms 86 6 Perspectives 88 7 References 90 8 Appendices 103 8.1 Supplementary Tables 103 8.2 Abbreviations 131 8.3 Publications 133 8.3.1 Journal articles 133 8.3.2 Oral presentations 133 8.3.3 Posters 134 8.4 Curriculum vitae 135 v vi Summary 1 1 Summary To become biologically active, proteins must reliably and rapidly acquire their cor- rect three-dimensional structures. It has been firmly established that all the folding informa- tion is contained within the amino acid sequence of a protein. However, the mechanisms utilized by proteins to avoid sampling of the extraordinarily large number of possible con- formations during their folding process are just beginning to be understood. In the cell, effi- cient folding of many newly synthesized proteins relies on the assistance of molecular chap- erones, which act to prevent aggregation and promote folding. In Escherichia coli (E. coli), nascent chain-binding chaperones, such as DnaK (a member of the Hsp70 family), stabilize elongating polypeptides on ribosomes and prevent them from aggregating. Folding of the complete nascent chain in the cytosol is achieved either upon controlled release from these factors and/or following transfer to downstream chaperones, such as the GroEL/GroES chaperonin. This complex is formed by the barrel-shaped GroEL oligomer (also called Hsp60) and its GroES cofactor (Hsp10), which functions as a detachable lid. Newly synthe- sized proteins encapsulated by the chaperonin can attain their native structure unimpaired by aggregation, during repeated cycles of ATP-dependent binding and release. Remarkably, in spite of our detailed knowledge of the chaperonin reaction mecha- nism, only little is known about the natural substrates of GroEL. Most mechanistic studies on the E. coli chaperonin have been performed with heterologous substrates. Our laboratory previously identified ~50 GroEL-interacting proteins (Houry et al., 1999), but their depend- ence on GroEL during the process of protein folding remained to be determined. In an at- tempt to define the requirement of a polypeptide for GroEL-assisted folding, the folding properties of ten GroEL-interacting proteins were studied both in vitro and in vivo. The development of in vitro refolding assays allowed categorization of GroEL inter- acting proteins into three distinct classes. Class I proteins are largely independent of chaper- ones but can improve their folding yield by general chaperone interactions. Class II proteins do not refold efficiently in the absence of chaperones, but can utilize both the DnaK and the GroEL/GroES systems.
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