title: Modulation of protein properties in living cells using nanobodies.
abstract: Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.
authors.affiliation: Gene Center, Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Kirchhofer
authors.forename: Jonas
authors.initials: J
authors.lastname: Helma
authors.forename: Katrin
authors.initials: K
authors.lastname: Schmidthals
authors.forename: Carina
authors.initials: C
authors.lastname: Frauer
authors.forename: Sheng
authors.initials: S
authors.lastname: Cui
authors.forename: Annette
authors.initials: A
authors.lastname: Karcher
authors.forename: Mireille
authors.initials: M
authors.lastname: Pellis
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Corella S
authors.initials: CS
authors.lastname: Casas-Delucchi
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Cardoso
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Karl-Peter
authors.initials: KP
authors.lastname: Hopfner
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002162:Camelus / Q000276:immunology*; D003460:Crystallization; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D000906:Antibodies; D049452:Green Fluorescent Proteins