Reference : VHHs as model proteins to investigate amyloid fibril formation: Fibril stability and eff...
Scientific congresses and symposiums : Poster
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/94743
VHHs as model proteins to investigate amyloid fibril formation: Fibril stability and effect of seeding and cross-seeding on aggregation kinectics
English
Chavignon, Chloé mailto [Université de Liège - ULg > > > Doct. sc. (bioch., biol. mol.&cell., bioinf.&mod.-Bologne)]
Jun-2010
No
Gordon research conference on Biolpoylmers
06-11.06.2010
Gordon research conference
Newport
RI
[en] VHH ; amyloid fibril
[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 amyloidosis are known so far, amongst which Alzheimer's disease, type II diabetes and immunoglobulin amyloidosis [1].

Although the mechanism of amyloid fibrils 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 no more restrained to proteins involved in amyloidosis but can be settled on a wide variety of proteins.

In this study, we have chosen to investigate the mechanism of amyloid fibrils formation by two variable domains of camelid heavy-chain antibodies (referred to as VHHs or nanobodies), cAb-HuL6 and cAb-BcII10, and this choice was motivated by the following reasons:
- First, VHHs are small monomeric proteins (~14 kDa) presenting a high stability and a high solubility [2], which permits their expression with a high yield (20-40 mg.L-1).
- Second, a wide range of stable mutants of these two VHHs is available. Mutations located at the disulfide bond [3,4], the CDRs [3] and the framework have been introduced. 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 intermediates and thus amyloid fibrils, heat-induced unfolding experiments at pHs comprised in a range from 2,5 to 9,5 have been monitored by intrinsic fluorescence, ANS binding 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 electronic microscopy. We will discuss the kinetics of aggregation obtained in the absence and the presence of seeding/cross-seeding.


[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
Etude du mécanisme moléculaire de la formation de fibres amyloïdes en utilisant des fragments d’anticorps à chaînes lourdes comme protéines modèles
http://hdl.handle.net/2268/94743

File(s) associated to this reference

Fulltext file(s):

FileCommentaryVersionSizeAccess
Restricted access
100606 - GRC.pdfPublisher postprint855.67 kBRequest copy

Bookmark and Share SFX Query

All documents in ORBi are protected by a user license.