25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Spin-labeled nanobodies as proteins’ conformational reporters towards in-cell EPR applications.

28 Aug 2019, 11:05
25m
Lecture Hall D (Henry Ford Building)

Lecture Hall D

Henry Ford Building

Talk EPR development and applications EPR

Speaker

Dr Laura Galazzo (Ruhr University Bochum)

Description

Nanobodies (i.e. single-domain antibodies) are promising new tools for in-cell applications due to their low molecular weight, protein- and state- specificity, nano- or sub-nano-molar affinity to their target and the possibility to be inserted into cells. We propose here the use of spin-labeled nanobodies as conformational reporters of wild type unlabeled proteins via DEER spectroscopy.
We focused on a set of spin-labeled nanobodies targeting ABC transporters, proteins that couple the energy deriving from the binding and hydrolysis of ATP to large conformational changes that enable substrate translocation across membranes. These molecular machines have been studied in different environments (as detergent, liposomes and nanodiscs) via different techniques[1,2]; in this framework, the challenge ahead is the investigation of their conformational plasticity in a native environment.
We show here[3] the applicability of the use of gadolinium-labeled nanobodies against the heterodimeric exporter TM287/288: thanks to a “cocktail” of state- and non-state-specific nanobodies binding to different sites of the protein, we were able to obtain a fingerpint distance of the outward-facing state of the transporter and follow the conformational cycle of the unlabeled wild type protein. Orthogonal labels attached to the transporter were also used to corroborate and strengthen the findings. With this proof of principle, we then used a non-state-specific nanobody showing high affinity for both nucleotide binding domains of the homodimeric exporter MsbA to gather structural information in detergent, proteoliposomes, nanodiscs and inside-out vesicles. Our results show a strong structural dependency on the environment, proving the need of an investigation in a native environment. Reliable structural information at low micromolar protein concentrations were obtained, paving the way for the use of biocompatible Gd-labeled nanobodies in cells.

[1] Mi, W. et al., 2017, Nature, 549, 233-237.
[2] Borbat, P.P. et al., 2007, PloS biology, 5, e271.
[3] Galazzo, L. et al., 2019, in preparation.

Primary authors

Dr Laura Galazzo (Ruhr University Bochum) Dr M. Hadi Timachi (Ruhr University Bochum) Mr Gianmarco Meier (Institute of Medical Microbiology, University of Zürich) Dr Cedric A.J. Hutter (Institute of Medical Microbiology, University of Zürich) Prof. Markus A. Seeger (Institute of Medical Microbiology, University of Zürich) Prof. Enrica Bordignon (Ruhr University Bochum)

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