Modulation of protein properties in living cells using nanobodies.
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.
Kirchhofer
Axel
A
Gene Center, Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany.
Helma
Jonas
J
Schmidthals
Katrin
K
Frauer
Carina
C
Cui
Sheng
S
Karcher
Annette
A
Pellis
Mireille
M
Muyldermans
Serge
S
Casas-Delucchi
Corella S
CS
Cardoso
M Cristina
MC
Leonhardt
Heinrich
H
Hopfner
Karl-Peter
KP
Rothbauer
Ulrich
U
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
D000906:Antibodies; D049452:Green Fluorescent Proteins