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Elongation of Actin Upon Binding to Prefoldin
Linköping University, Department of Physics, Chemistry and Biology.
(English)Manuscript (Other (popular scientific, debate etc.))
Abstract [en]

This pilot study on the interaction between actin andprefoldin indicates that actin binds to prefoldin in astretched conformation, probably similar to the bindingconformation at the initial interaction with TRiC/ADP as shown in a previous study (8). Further refinement of theprefoldin purification is a future aim, as well as additionalhomo-FRET measurements on several actin variantsspread throughout the structure to investigate the localvolume expansions/compressions of the prefoldin-boundactin molecule and compare the results to actin bound tothe chaperonins TRiC/ADP, TRiC/AMP-PNP and GroEL(8, 18). A fairly detailed comparison between thedistances throughout the actin molecule when bound toprefoldin and TRiC/ADP could reveal if the stretching inprefoldin is the same as in TRiC/ADP, or if there isadditional effects on the actin molecule as it is beingtransferred from prefoldin to the TRiC cavity.

National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-13127OAI: diva2:17876
Available from: 2008-04-03 Created: 2008-04-03
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.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1099
Biochemistry, chaperonin mechanisms, bound target protein, fluorescence resonance energy transfer (FRET), Actin, abundant eukaryotic protein
National Category
Biochemistry and Molecular Biology
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)
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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