Reference : VHHs as model proteins to investigate amyloid fibril formation: effect of seeding and cr...
Scientific congresses and symposiums : Poster
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/103999
VHHs as model proteins to investigate amyloid fibril formation: effect of seeding and cross-seeding on the stability of fibrils
English
Chavignon, Chloé mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Pardon, Els []
Wyns, Lode []
Muyldermans, Serge []
Dumoulin, Mireille mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Aug-2011
A0
No
No
International
Amyloid 2011 - Amyloid fibrils, prions and precursors: Molecules for targeted intervention
du 25 au 28 août 2011
Marcus Fändrich
Halle
Allemagne
[en] amyloid fibrils ; camelid heavy-chain antibody (VHH)
[en] The term "amyloidosis" covers a group of diseases associated with the deposition of protein aggregates organized into amyloid fibrils in different organs. About forty amyloidoses are known so far, amongst which Alzheimer's disease, type II diabetes and immunoglobulin amyloidosis [1]. Although the mechanism of amyloid fibril formation at the molecular level is not yet completely understood, it has been shown that the capacity to form amyloid fibrils in vitro is an intrinsic property of all polypeptide chains [1]. The choice of model proteins to investigate the aggregation process in vitro is therefore not restrained to proteins involved in amyloidoses but can be settled on a wide variety of proteins.
In this study, we have chosen to investigate the mechanism of amyloid fibril formation by two variable domains of camelid heavy-chain antibodies (referred to as VHHs or nanobodies), cAb-HuL6 and cAb-BcII10, for which variants with mutations located at the disulfide bond [3,4] and the CDRs [3] are available. Characterisation of the aggregating properties of these mutants will allow the investigation of the impact of these structural elements on the process of fibril formation.
In order to determine conditions in which cAb-HuL6 and cAb-BcII10 are more susceptible to form amyloid fibrils, heat-induced unfolding experiments at several pHs have been monitored by intrinsic fluorescence and circular dichroism. Then, aggregation experiments have been performed in the selected conditions and the presence of amyloid fibrils has been acknowledged by thioflavineT fluorescence experiments and electron microscopy. We will discuss the kinetics of aggregation obtained in the absence and the presence of seeding/cross-seeding and the stability of the formed fibrils.

[1] Chiti and Dobson, Annu. Rev. Biochem., 75, 2006, 333-366 ; [2] Dumoulin et al., Protein Sci., 11, 2002, 500-515 ; [3] Saerens et al., J. Mol. Biol., 352, 2005, 597-607 ; [4] Saerens et al., J. Mol. Biol., 377, 2008, 478-488.
Centre d'Ingénierie des Protéines - CIP
Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture (Communauté française de Belgique) - FRIA ; Fonds de la Recherche Scientifique (Communauté française de Belgique) - F.R.S.-FNRS
Etude du mécanisme moléculaire de la formation de fibres amyloïdes à l'aide de fragments d'anticorps à chaînes lourdes comme protéines modèles
Researchers ; Students
http://hdl.handle.net/2268/103999

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