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Interactions Between the Bacterial β-actin Homologue MreB and the Group I Chaperonin GroEL and Group II Chaperonin TRiC
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.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

This pilot study on the interaction between MreB andthe chaperonins TRiC and GroEL indicates that thefolding of the actin ancestor was facilitated by thechaperonins. From an evolutionary point of view, it isinteresting to investigate the nature of the bindinginteraction between the prokaryotic system MreB-GroELand compare it to the binding interaction between actinand TRiC and the following questions will be addressed:Does MreB refold in a spontaneous manner or is itsfolding dependent on the action of a chaperonin (GroEL)as in the case of actin folding (TRiC)? Does MreB bind ina similar stretched manner to GroEL as actin binds toTRiC (4, 11), or is the “general” binding inducedunfolding sufficient for guiding MreB to the native state?How does the MreB molecule interact with TRiC, is therea similar stretching as for actin? Are there any analoguessequences between actin and TRiC that are recognized byTRiC and/or GroEL?

Two single variants where cysteines have beenintroduced at positions 69 and 245 in E. coli MreB(Figure 1 B). These positions are situated at the tips of thecorresponding subdomains 2 and 4 of the actin molecule(4, 12). The double variant N69C/V245C has also beenconstructed. The three variants will be produced andlabeled with fluorescein and subsequent homo-FRETmeasurements will be performed on MreB bound toGroEL, TRiC and GroES. The results will be comparedto the results on actin bound to the chaperonins toinvestigate how the chaperonin-dependent folding ofactin homologues has evolved.

National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-52935OAI: oai:DiVA.org:liu-52935DiVA: diva2:285951
Available from: 2010-01-13 Created: 2010-01-13 Last updated: 2010-01-13Bibliographically approved
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, LailaMoparthi, Satish BabuHammarström, PerJonsson, Bengt-Harald

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