Reference : Engineering a camelid antibody fragment that binds to the active site of human lysozyme ...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/22376
Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils
English
Chan, Pak Ho [University of Cambridge > Department of Chemistry > > >]
Pardon, Els [Vrije Universiteit Brussel - VUB > > > >]
Menzer, Linda [Université de Liège - ULg > > > >]
De Genst, Erwin [University of Cambridge > > > >]
Kumita, Janet [University of Cambridge > > > >]
Christodoulou, John [University of Cambridge > > > >]
Saerens, Dirk [> >]
Brans, Alain mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines > > >]
Bouillenne, Fabrice mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines >]
Archer, David [University of Nottingham > > > >]
Robinson, Carol [University of Cambridge > > > >]
Muyldermans, Serge [Vrije Universiteit Brussel - VUB > > > >]
Matagne, André mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines, Centre d'Ingénierie des Protéines > > >]
Redfield, Christina [University of Oxford > > > >]
Wyns, Lode [Vrije Universiteit Brussel - VUB > > > >]
Dobson, Christopher M. [> >]
Dumoulin, Mireille mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines, Centre d'Ingénierie des Protéines > >]
2008
Biochemistry
American Chemical Society
47
11041-11054
Yes (verified by ORBi)
International
0006-2960
1520-4995
Washington
DC
[en] lysozyme ; nanobodies ; amyloid fibril ; H/D exchange ; NMR ; mass spectrometry ; misfolding ; camelid antibody
[en] single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation.
http://hdl.handle.net/2268/22376
also: http://hdl.handle.net/2268/129908

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