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Structural Rearrangements of Actins Interacting with the Chaperonin Systems Tric/Prefoldin and Groel/ES ______

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Structural Rearrangements of Actins Interacting with the Chaperonin Systems Tric/Prefoldin and Groel/ES ______ Structural Rearrangements of Actins Interacting with the Chaperonin Systems TRiC/Prefoldin and GroEL/ES _____________________________________________________________________ Linköping Studies in Science and Technology Dissertation No. 1099 Structural Rearrangements of Actins Interacting with the Chaperonin Systems TRiC/Prefoldin and GroEL/ES Laila Villebeck Molecular Biotechnology Department of Physics, Chemistry and Biology Linköping University, SE-581 83 Linköping, Sweden Linköping 2007 On the cover: Close-up of the E-actin structure © 2007 Laila Villebeck ISBN: 978-91-85715-05-3 ISSN: 0345-7524 Printed in Sweden by LiU-Tryck Linköping 2007 “Impossible is just a big word thrown around by small men who find it easier to live in the world they’ve been given than to explore the power they have to change it. Impossible is not a fact. It’s an opinion. Impossible is not a declaration. It’s a dare. Impossible is potential. Impossible is temporary. Impossible is nothing.” - Adidas Advertising ABSTRACT The studies in this thesis are mainly focused on the effects that the chaperonin mechanisms have on a bound target protein. Earlier studies have shown that the bacterial chaperonin GroEL plays an active role in unfolding a target protein during the initial binding. Here, the effects of the eukaryotic chaperonin TRiC’s mechanical action on a bound target protein were studied by fluorescence resonance energy transfer (FRET) measurements by attaching the fluorophore fluorescein to specific positions in the structure of the target protein, E-actin. Actin is an abundant eukaryotic protein and is dependent on TRiC to reach its native state. It was found that at the initial binding to TRiC, the actin structure is stretched, particularly across the nucleotide-binding site. This finding led to the conclusion that the binding-induced unfolding mechanism is conserved through evolution. Further studies indicated that in a subsequent step of the chaperonin cycle, the actin molecule collapses. This collapse leads to rearrangements of the structure at the nucleotide-binding cleft, which is also narrowed as a consequence. As a comparison to the productive folding of actin in the TRiC chaperonin system, FRET studies were also performed on actin interacting with GroEL. This is a non- productive interaction in terms of guiding actin to its native state. The study presents data indicating that the nucleotide-binding cleft in actin is not rearranged by GroEL in the same way as it is rearranged during the TRiC interaction. Thus, it could be concluded that although the general unfolding mechanism is conserved through the evolution of the chaperonins, an additional and specific binding to distinct parts of the actin molecule has evolved in TRiC. This specific binding leads to a directed unfolding and rearrangement of the nucleotide-binding cleft, which is vital for actin to reach its native state. The differences in the chemical properties of the actin-GroEL and the actin-TRiC complexes were also determined by measurements of fluorescein anisotropies and AEDANS emission shifts for probes attached to positions spread throughout the actin structure. The evolutionary aspects of the chaperonin mechanisms and the target protein binding were further investigated in another study. In this study, the prokaryotic homologue to actin, MreB, was shown to bind to both TRiC and GroEL. MreB was also shown to bind to the co-chaperonin GroES. In a separate study, the interaction between actin and the chaperone prefoldin was investigated. In vivo prefoldin interacts with non-native actin and transfers it to TRiC for subsequent and proper folding. In this homo-FRET study, it was shown that actin binds to prefoldin in a stretched conformation, similar to the initial binding of actin to TRiC. vii viii INCLUDED PAPERS This thesis is based on the results from three scientific papers and two progress reports, which are listed below and enclosed at the end of the thesis. They will be referred to in the text by the roman numerals (Papers I-III and Progress reports I-II): Paper I: Conformational Rearrangements of Tail-less Complex Polypeptide 1 (TCP- 1) Ring Complex (TRiC)-Bound Actin Laila Villebeck, Malin Persson, Shi-Lu Luan, Per Hammarström, Mikael Lindgren, and Bengt-Harald Jonsson Biochemistry, 2007, In press Paper II: Different Conformational Effects when β-actin Binds to the Bacterial Chaperonin GroEL and the Eukaryotic Chaperonin TRiC Laila Villebeck, Satish Babu Moparthi, Mikael Lindgren, Per Hammarström, and Bengt-Harald Jonsson Submitted Paper III: Mapping the Different Interactions Between the Eukaryotic β-actin and Group I (GroEL) and Group II (TRiC) Chaperonins Laila Villebeck, Hanna Klang, Satish Babu Moparthi, Mikael Lindgren, Per Hammarström, and Bengt-Harald Jonsson In manuscript Progress report I: Interactions Between the Bacterial β-actin Homologue MreB and the Group I Chaperonin GroEL and Group II Chaperonin TRiC Laila Villebeck, Satish Babu Moparthi, Per Hammarström, and Bengt-Harald Jonsson In manuscript Progress report II: Elongation of Actin Upon Binding to Prefoldin Laila Villebeck, Sara Johansson, and Bengt-Harald Jonsson In manuscript ix Contribution report Paper I: I participated in the planning process, performed some of the mutagenisis to create the actin variants, performed all steady-state fluorescence experiments, analyzed the data, and did a major part of the writing. I also took part in supervising Maria Falklöf during her undergraduate thesis work. Paper II: I planned the project, performed all steady-state fluorescence experiments, analyzed the data, and did a major part of the writing. Paper III: I planned the project, performed the steady-state and time-resolved fluorescence measurements on the GroEL samples together with Hanna Klang and Mikael Lindgren. I also performed all of the steady-state fluorescence measurements on the TRiC samples. I analyzed the steady-state data and did a major part of the writing. I was also Hanna Klang’s supervisor during her undergraduate thesis work. Progress report I: I planned the project, performed the cloning experiments and most of the mutagenisis to create the MreB variants. I performed all fluorescence experiments, analyzed the data, and did a major part of the writing. Progress report II: I planned the project, developed the prefoldin purification procedure together with Sara Johansson, who I also supervised during her undergraduate thesis work. I did all of the fluorescence experiments that are included in this report, analyzed the data and did a major part of the writing. x POPULÄRVETENSKAPLIG SAMMANFATTNING I denna avhandling presenteras resultat från studier på hur chaperoner påverkar proteiner som sitter bundna till dem. Proteiner har viktiga uppgifter i våra celler där de sköter bland annat energiförsörjning, nedbrytning av slaggprodukter, transport av näringsämnen och andra makromolekyler, upptag av syre i lungorna, kommunikation mellan olika organ i kroppen, försvar mot virus och bakterier med mera. Proteiner är beroende av sin tredimensionella struktur för att fungera. I cellen finns många processer som gör att proteinerna förlorar sin struktur och det är här chaperonerna kommer in i bilden. Chaperoner är ”hjälparmolekyler”, oftast är de själva proteiner, som finns i våra celler där de bryter och förhindrar bildandet av oönskade interaktioner i och mellan proteiner. De chaperoner som varit av det huvudsakliga intresset i denna avhandling tillhör en särskild familj av chaperoner som kallas chaperoniner. Chaperoninerna är formade som tunnor. Inuti tunnan kan proteiner med felaktig struktur binda och finna sin rätta struktur. Den exakta mekanismen för hur detta går till är ännu inte klarlagt, vilket gör detta till ett spännande forskningsområde. Studierna som presenteras i avhandlingen visar att när proteinet aktin, som finns i eukaryota celler till exempel däggdjursceller, binder till chaperoninen TRiC så sträcks aktinet ut vilket leder till att delar av strukturen arrangeras om. I ett senare steg i chaperoninens cykliska mekanism kompakteras aktinstrukturen. Denna mekanism med utsträckning följt av kompaktering har av andra forskningsgrupper också presenterats för den bakteriella chaperoninen GroEL vilket ledde till slutsatsen att chaperoninernas mekanism är bevarad genom miljontals år av evolution. GroEL kan dock inte hjälpa aktin att hitta sin rätta struktur trots att den allmänna mekanismen är liknande den som finns i TRiC. Här presenteras resultat som visar att aktin binder in annorlunda till GroEL än till TRiC och att det stora omarrangemang av strukturen som observerades i TRiC inte sker i GroEL. Detta resultat ledde till slutsatsen att bortsett från den allmänna sträckningsmekanismen som är likadan för TRiC och GroEL, så har TRiC under evolutionen utvecklat en specific igenkänning och inbindning av aktin. Den specifika igenkänningen leder till att en del av strukturen som inte kan hitta rätt av sig självt kan guidas rätt. Avhandlingen innehåller också bland annat en förstudie som visar att aktinets motsvarighet i bakterier, MreB, binder till båda chaperoninerna TRiC och GroEL samt även till en co-chaperonin till GroEL som kallas GroES. Ytterligare en annan studie visar resultat på att aktin binder in i utsträckt form till ett annat chaperon, prefoldin. Prefoldin samarbetar med TRiC i cellen genom att fånga upp proteiner med fel struktur och lämna över dem till TRiC för vidare guidning till rätt struktur. xi xii ACKNOWLEDGEMENTS Som barn har man många drömmar och de växlar med tiden. När jag var liten ville jag bli veterinär, jobba med hästar, bli forskare eller författare. Jag var extremt blyg och ville ha ett yrke där man inte har så mycket kontakt med människor. Jag var också rädd för att göra fel. Särskilt ifall det fanns folk runtomkring som såg att jag gjorde fel… Min bild av en forskare då var en enstöring som satt ensam på sin kammare och jobbade hårt för att lösa något av livets alla mysterier. Perfekt tyckte jag! - Hahaha! Vilken tur att jag hade fel att man inte skulle behöva träffa så många människor om man blev forskare! Och vilken tur att man faktiskt får göra fel och att det till och med kan vara rätt att göra fel ibland.
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