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.

HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

“…1D). Although V H H-8 and V H H-1 represent the most similar V H H pair, V H H-9 is the most distant representative based on sequence homology analysis (26).…”

“…E. coli) or eukaryotic cells without loss of their antigen recognition properties (25). V H Hs are easily modified and can be equipped with fluorescent or affinity tags to track their target antigens within the cell (26,27). V H Hs can serve as specific and efficient protein inhibitors or modulators and can induce changes in protein conformation within cells.…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…105 By contrast, sdAb paratopes can clearly adopt both flat 106,107 and convex 11 topologies, although possibly only inefficiently adopt concave ones. The CDR1 and CDR2 loops of V H Hs depart from the typical canonical structures of conventional antibodies (Figure 2A), potentially through somatic mutation since germline human V H and camelid V H H repertoires appear to have similar canonical structures.…”

Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments

“…a B2H selection, to retrieve functional variants. The BM_GFP2 and BM_GFP3 were selected previously by a B2H against GFP (42). Both binders have an additional Cys residue in CDR1 at position 38 and another Cys residue in CDR3 at positions 112.5 and 111.3 for BM_GFP2 and BM_GFP3, respectively.…”