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Modulation of protein properties in living cells using nanobodies
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Modulation of protein properties in living cells using nanobodies
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Journal Article
Modulation of protein properties in living cells using nanobodies
2010
Overview
GFP fluorescence can be modulated in mammalian cells by binding to single-chain antibodies (nanobodies), selected to make GFP brighter or dimmer; these changes are explained by the crystal structures of the GFP-nanobody complexes. The applications of such nanobodies to monitor protein expression and subcellular localization in real time are explored.
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.
Publisher
Nature Publishing Group US,Nature Publishing Group
