Positive Functional Synergy of Structurally Integrated, Designed Artificial Protein Dimers Assembled by Fully Genetically Encoded Click Chemistry

We combined <i>in silico</i>modelling with fully genetically encoded strain promoted azide-alkyne cycloaddition, to construct bespoke protein dimers. Using fluorescent proteins GFP and Venus as models, homo and heterodimers were constructed that switched ON once assembled and displayed enhanced spectral properties. The determined molecular structure reveals long range polar bond networks involving amino acids and structured water molecules play a key role in activation and functional enhancement by directly linking the two functional centres. Single molecule analysis revealed the dimer is more resistant to photobleaching spending longer times in the ON state with only one CRO likely to be active at any one time. Thus, genetically encoded bioorthogonal chemistry can be used beyond simple passive linkage approaches to generate new and truly integrated protein complexes that form long range bonds networks, which have a profound effect on function and our understanding of fluorescent protein function.