A new thermostable fluorescent protein is shown to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.
Protein-fragment complementation
assays are used ubiquitously for
probing protein–protein interactions. Most commonly, the reporter
protein is split in two parts, which are then fused to the proteins
of interest and can reassemble and provide a readout if the proteins
of interest interact with each other. The currently known split fluorescent
proteins either can be used only in aerobic conditions and assemble
irreversibly, or require addition of exogenous chromophores, which
complicates the design of experiments. In recent years, light-oxygen-voltage
(LOV) domains of several photoreceptor proteins have been developed
into flavin-based fluorescent proteins (FbFPs) that, under some circumstances,
can outperform commonly used fluorescent proteins such as GFP. Here,
we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing
protein from Chloroflexus aggregans, can serve as
a split fluorescent reporter. We use the available genetic and structural
information to identify three loops between the conserved secondary
structure elements, Aβ-Bβ, Eα-Fα, and Hβ-Iβ,
that tolerate insertion of flexible poly-Gly/Ser segments and eventually
splitting. We demonstrate that the designed split pairs, when fused
to interacting proteins, are fluorescent in vivo in E. coli and human cells and have low background fluorescence.
Our results enable probing protein–protein interactions in
anaerobic conditions without using exogenous fluorophores and provide
a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based
fluorescent reporters and optogenetic tools.
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