Malachitgrüner konjugierter Antikörper

We report that a symmetric small molecule ligand mediates assembly of the antibody light chain variable domains (V  _L  s) into a corresponding symmetric ternary complex with novel interfaces. L5* Fluorogen Activating Protein (FAP) is a VL  _domain  that binds malachite green (MG) dye to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with extensive C2 symmetry, with MG almost completely encapsulated between an antiparallel arrangement of the two  _VL  domains. _Unliganded L5*   VL domains crystallize as a similar antiparallel  _VL /V  _L  homodimer. The complementarity determining regions (CDRs) are spatially aligned to form new V _L  /V  _L  and V  _L  /ligand interfaces that tightly constrain a propeller conformer of MG. 

Binding equilibrium analysis suggests a highly cooperative assembly to form a very stable V  _L  /MG/V  _L  complex such that MG behaves as a strong chemical inducer of dimerization. Fusion of two V  _LDomains to a single protein tightens MG binding more than 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that binding interactions with ligands and the restriction of domain motions make independent contributions to the binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes in which unnatural symmetrical binding sites are preferred over canonical antibody sites. As exemplified by L5*, these self-reporting complexes can be useful as modulators of protein assembly or as high-affinity protein tags and capture reagents.

introduction

The classical vertebrate immune system is capable of raising antibodies that specifically recognize and bind to a very diverse repertoire of immunogens, including natural and synthetic small molecules. _{The variable domains of the heavy (VH}  ) and light chain (V  _L  ) of immunoglobulin (IgG) form a heterologous binding pocket where typically amino acids from both domains contact the small molecule ligand. 

• Isolated V  _H  and V  _L domains bind  to each other along an evolutionarily conserved cleavage site found in parent IgG, forming a binding cleft flanked at its open end by six protein backbone loops, three from each domain. ¹
• Molecular recognition abilities are largely governed by these loops, termed  c complementarity determining  regions (CDRs), which undergo somatic hypermutation during the immune response to produce specific high-affinity binding to the antigen  . The V  _H  and V  _L  domains can be genetically fused via short peptide linkers to generate single chain antibodies (scFvs) that generally retain the architecture and binding functionality of the IgG variable region.
• We have used scFvs as a platform to develop fluorescent biosensors and reporters for use in living systems .
• The technology is based on certain organic dyes (fluorogens) that are non-fluorescent when free in solution but become intensely fluorescent when specifically bound by related scFvs (fluorogen-activating proteins or FAPs) .
• The scFv binding pocket is thought to physically restrict rotation around the methine bonds of the dye chromophore, thereby promoting fluorescence emission rather than non-radiative depletion. FAPs are easily engineered and expressed as fusions with proteins of interest. Biological targets in living cells can be specifically visualized by the addition of fluorogens without the need for washing procedures, since unbound fluorogens do not fluoresce. visualization of targets in cells, on cell surface,  or within vesicle,  can be selectively controlled using membrane-permeable or impermeable fluorogens.
• Several apparent scFvs, each activating several different methine dyes, were isolated from a yeast surface display library expressing ~10  ⁹  recombinantly fused V  _H  V  _L  domains derived from human spleen and lymph node cDNAs; small subpopulations of single V  _H  and V  _L  domains are also expressed. ScFv/methine dye complexes that fluoresce in the near UV,  visible  and far red  Spectral ranges were characterized.
• In addition to enhancing fluorescence, binding to related scFvs modulates the excitation and emission maxima of these methine dyes, producing 5–60 nm red-shifts relative to unbound dye.

Six proteins isolated from the library specifically activate a pegylated derivative of malachite green (MG).

 MG-binding FAPs amplify fluorescence up to 20,000-fold compared to unbound dye to brightness levels typical of fluorescent proteins and emit in the far-red spectral region (~670 nm) where cellular autofluorescence is low, making these fluorine modules well suited are for further development. Although isolated from a nominal scFv library, five of these proteins function efficiently as FAP when expressed from just a single heavy or light variable domain gene  but the stoichiometry and architecture of the functional FAP/MG complexes remain unknown. To understand the 3D structure of these unusual immune protein/ligand complexes and the detailed mechanism by which malachite green binds and fluoresces, we determined their crystallographic structures and studied their physical and biochemical properties.

Here we report the structure and properties of a spectrally enhanced FAP carrying a point mutation generated during affinity maturation of the precursor L5 V  _L  ³  . We examine the properties of a genetically fused tandem dimer of this VL  _and  a tandem dimer of a VL  _carrying  a second mutation that improves binding affinity ( Fig. 1b ). Figure 1a presents the Kabat numbering of the relevant mutations, and Table 1lists the simplified nomenclature we use in this paper, along with cross-references to previous studies that used some of these improved FAPs but with different nomenclature. Improved versions of L5 fluorine modules have so far been used  in vivo  for stimulated emission depletion (STED) super-resolution microscopy, for a fluorescence resonance energy transfer system that amplifies the FAP signal, as a  _VL  component in hybrid V H  _{VL  –}  Sensors applied , as a sensor of protein O-glycosylation,  and to trafficking studies of anions – and cations – channels.