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Structural rearrangements of actins interacting with the Chaperonin systems TRiC/Prefoldin and GroEL/ES
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
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 [en]
Biochemistry, chaperonin mechanisms, bound target protein, fluorescence resonance energy transfer (FRET), Actin, abundant eukaryotic protein
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-11445ISBN: 978-91-85715-05-3 (print)OAI: oai:DiVA.org:liu-11445DiVA: diva2:17877
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
List of papers
1. Conformational Rearrangements of Tail-less Complex Polypeptide 1 (TCP-1) Ring Complex (TRiC)-Bound Actin
Open this publication in new window or tab >>Conformational Rearrangements of Tail-less Complex Polypeptide 1 (TCP-1) Ring Complex (TRiC)-Bound Actin
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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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13123 (URN)10.1021/bi062093o (DOI)
Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2009-05-18
2. Different Conformational Effects when β-actin Binds to the Bacterial Chaperonin GroEL and the Eukaryotic Chaperonin TRiC
Open this publication in new window or tab >>Different Conformational Effects when β-actin Binds to the Bacterial Chaperonin GroEL and the Eukaryotic Chaperonin TRiC
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2007 (English)Article in journal (Refereed) Submitted
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13124 (URN)
Available from: 2008-04-03 Created: 2008-04-03
3. Mapping the Different Interactions between Eukaryotic β-actin and the Group I (GroEL) and Group II (TRiC) Chaperonins
Open this publication in new window or tab >>Mapping the Different Interactions between Eukaryotic β-actin and the Group I (GroEL) and Group II (TRiC) Chaperonins
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Productive folding to the native state of the abundant eukaryotic protein actin is dependent on the chaperonin TRiC. The prokaryotic chaperonin GroEL also recognizes actin, but this interaction does not lead to the correct folding of actin. It is well established that GroEL interacts with non-native proteins through hydrophobic interactions. The characteristics of the interactions between TRiC and its target proteins are however unclear. In this study, we present multiple site-directed cysteine labeling and fluorescence measurements indicating that actin initially binds to TRiC through several interaction sites and that the surfaces of the interaction areas on the walls of the TRiC chamber present both polar and hydrophobic residues. At a later stage in the chaperonin cycle, the binding of ATP causes conformational changes in the chaperonin, which leads to a presentation of a more hydrophobic milieu in TRiC chamber. The conformational changes of the chaperonin causes rearrangements of the actin molecule and new interactions are proposed to be formed. Additionally, we show that the initial binding of actin to TRiC leads to a re-modeling of the nucleotide-binding cleft in actin. We also present data indicating that GroEL presents less specific interaction areas towards the bound actin than TRiC does. The interactions between actin and GroEL are tight and of hydrophobic character. No re-modeling of the nucleotide-binding cleft was obtained in the actin-GroEL complex. We conclude that the interactions between actin and TRiC are of both polar and hydrophobic character, the nature of the interactions are different in the prokaryotic and eukaryotic chaperonins, and the rearrangements of the nucleotide binding cleft of actin seen in the chaperonin cycle of TRiC do not occur in GroEL. We suggest that the rearrangements of the nucleotide-binding site in actin are critical for productive folding of actin.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-52934 (URN)
Available from: 2010-01-13 Created: 2010-01-13 Last updated: 2010-01-13
4. Interactions Between the Bacterial β-actin Homologue MreB and the Group I Chaperonin GroEL and Group II Chaperonin TRiC
Open this publication in new window or tab >>Interactions Between the Bacterial β-actin Homologue MreB and the Group I Chaperonin GroEL and Group II Chaperonin TRiC
(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:nbn:se:liu:diva-52935 (URN)
Available from: 2010-01-13 Created: 2010-01-13 Last updated: 2010-01-13Bibliographically approved
5. Elongation of Actin Upon Binding to Prefoldin
Open this publication in new window or tab >>Elongation of Actin Upon Binding to Prefoldin
(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
Identifiers
urn:nbn:se:liu:diva-13127 (URN)
Available from: 2008-04-03 Created: 2008-04-03

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