New applications in living cells
Antibodies are specialized proteins that mark foreign substances as targets for an immunological attack. This makes them an ideal tool for research and therapy because they can bind specifically to almost any chemical structure. Conventional antibodies, though, are very large and tend to clump in living cells. Camels and their South American relatives (alpacas, guanacos, llamas, vicuñas), however, also produce antibodies that are considerably smaller. The recognition domains of these structures form the basis of so-called nanobodies which retain their binding activity within cells.
To obtain nanobodies specific for GFP, the LMU team and their colleagues from the TU Darmstadt, the Free University of Brussels and the LMU spin-off ChromoTek first immunized alpacas with GFP, then transferred the genetic information that corresponded to antibodies – including those specific for GFP – into bacteria. As Dr. Ulrich Rothbauer of ChromoTek explains, “These antibody fragments were synthesized by bacteria and could be tested for the ability to bind GFP. Seven such nanobodies were identified from a large nanobody library.”
GFP is shaped like a barrel, open at both ends, and the light-absorbing structure necessary for fluorescence – the chromophore – forms spontaneously inside the barrel. Absorption of light leads to green fluorescence, with the response depending on the exact conformation of the protein. Two of the nanobodies had marked effects on the signal emitted by isolated GFP. “Binding of one enhanced fluorescence fivefold, the other reduced it by fourfold, essentially allowing us to turn the signal on or off”, says Rothbauer. How this is achieved was revealed by structural studies at the Gene Center: “Our structural studies of the bound complexes showed that one nanobody pushed a particular region of the protein closer to the chromophore, while the other tilted it away” explains Axel Kirchhofer, the first author of the study.
To determine whether the enhancing nanobody could act as a sensor for GFP-linked proteins in cells, the researchers created cells that synthesized a hormone receptor tagged with GFP in the cytoplasm and expressed the enhancer on the inner face of the nuclear membrane. Addition of the hormone causes the receptor to move into the nucleus. “We were able to follow the process by measuring the fluorescence induced when the GFP tag was captured by the nanobody. This very successful collaboration between cell and structural biologist demonstrate that nanobodies recognize, induce and stabilize alternative protein conformations and that they can be used to study their functional significance in vivo”, says Rothbauer. (PH/suwe)
“Modulation of protein properties in living cells using nanobodies”
Axel Kirchhofer, Jonas Helma, Katrin Schmidthals, Carina Frauer, Sheng Cui, Annette Karcher, Mireille Pellis, Serge Muyldermans, Corella Casas Delucchi, M. Cristina Cardoso, Heinrich Leonhardt, Karl-Peter Hopfner and Ulrich Rothbauer
Nature Structural and Molecular Biology, 13 December
Dr. Ulrich Rothbauer
Biocenter at LMU Munich and ChromoTek GmbH
Phone: +49 (0) 89 / 2180-74287
Prof. Dr. Karl-Peter Hopfner
Department Chemistry and Biochemistry
Phone +49 (0) 89 / 2180-76953