Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages.

TitleNanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages.
Publication TypeJournal Article
Year of Publication2013
AuthorsGuilliams, T., F. El-Turk, A. K. Buell, E. M. O'Day, F. A. Aprile, E. K. Esbjörner, M. Vendruscolo, N. Cremades, E. Pardon, L. Wyns, M. E. Welland, J. Steyaert, J. Christodoulou, C. M. Dobson, and E. De Genst
JournalJ Mol Biol
Volume425
Issue14
Pagination2397-411
Date Published2013 Jul 24
ISSN1089-8638
Keywordsalpha-Synuclein, Calorimetry, Circular Dichroism, Epitope Mapping, Kinetics, Magnetic Resonance Spectroscopy, Protein Binding, Protein Denaturation, Protein Multimerization, Single-Domain Antibodies, Thermodynamics
Abstract

Nanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric αSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble αSyn, comprising approximately amino acids 118-131 and 137-140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when αSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of αSyn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation.

DOI10.1016/j.jmb.2013.01.040
Alternate JournalJ. Mol. Biol.
PubMed ID23557833
Grant ListMRC G1002272 / / Medical Research Council / United Kingdom
/ / Wellcome Trust / United Kingdom