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.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

HighlightsAn alpaca-derived γ-H2AX nanobody was generated.γ-H2AX chromobody was able to bind and precipitate phosphorylated H2AX peptide.γ-H2AX chromobody could be produced in bacterial as well as mammalian cells.Alternative epitope recognition by γ-H2AX chromobody was induced by ectopic XRCC1.Accessibility of γ-H2AX chromobody was hindered by MDC1 masking in vivo.

The understanding of cellular processes and their pathophysiological alterations requires comprehensive data on the abundance, distribution, modification, and interaction of all cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not about endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications, and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (VHH) from Camelidae. These extremely stable VHH domains can be produced in bacteria, coupled to matrices, and used for affinity purification and proteome studies. Alternatively, these VHH domains can be fused with fluorescent proteins and expressed in living cells. These fluorescent antigen-binding proteins called “chromobodies” can be used to detect and trace proteins and other cellular components in vivo. Chromobodies can, in principle, detect any antigenic structure, including posttranslational modifications, and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analysis. Depending on the epitope chosen, chromobodies can also be used to modulate protein function in living cells.FigureDetection of the nuclear lamina with lamin chromobody in living cells.