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Conformational Rearrangements of Tail-less Complex Polypeptide 1 (TCP-1) Ring Complex (TRiC)-Bound Actin
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Department of Clinical Microbiology, Umeå University, Umeå, Sweden.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
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2007 (English)In: Biochemistry, ISSN 0006-2960, Vol. 46, no 17, 5083-5093 p.Article in journal (Refereed) Published
Abstract [en]

The mechanism of chaperonins is still under intense investigation. Earlier studies by others and us on the bacterial chaperonin GroEL points to an active role of chaperonins in unfolding the target protein during initial binding. Here, a natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] was investigated to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. Using fluorescence resonance energy transfer (FRET) measurements on four distinct doubly fluorescein-labeled variants of actin, we have obtained a fairly detailed map of the structural rearrangements that occur during the TRiC−actin interaction. The results clearly show that TRiC has an active role in rearranging the bound actin molecule. The target is stretched as a consequence of binding to TRiC and further rearranged in a second step as a consequence of ATP binding; i.e., the mechanism of chaperonins is conserved during evolution.

Place, publisher, year, edition, pages
2007. Vol. 46, no 17, 5083-5093 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-13123DOI: 10.1021/bi062093oOAI: oai:DiVA.org:liu-13123DiVA: diva2:17872
Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2009-05-18
In thesis
1. Structural rearrangements of actins interacting with the Chaperonin systems TRiC/Prefoldin and GroEL/ES
Open this publication in new window or tab >>Structural rearrangements of actins interacting with the Chaperonin systems TRiC/Prefoldin and GroEL/ES
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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, β-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.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi, 2007. 72 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1099
Keyword
Biochemistry, chaperonin mechanisms, bound target protein, fluorescence resonance energy transfer (FRET), Actin, abundant eukaryotic protein
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-11445 (URN)978-91-85715-05-3 (ISBN)
Public defence
2007-05-25, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 14:00 (English)
Opponent
Supervisors
Note
On the day of the defence date the satus of article I was: In press.Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2010-01-13Bibliographically approved

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Villebeck, LailaPersson, MalinHammarström, PerJonsson, Bengt-Harald

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