Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation

Abstract

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging.

Stabilization of structure in near-infrared fluorescent proteins by binding of biliverdin chromophore

“…The structure of NIR FP variants with Cys15 in the PAS domains may be additionally fastened by the threading of BV-containing N-terminal part of the protein through the knot. The importance of the knot for interaction with BV is indicated by tolerance of iRFP713 to a circular permutation mainly at N-terminal tail of PAS domain and at unstructured loop between PAS and GAF domains, but not at knotforming loop of GAF-domain [54].…”

Near-Infrared Fluorescent Proteins and Their Applications

“…However, it must be taken into account that BphP contains an unusual structural element, figure of eight knot, formed by the N terminal part of the PAS domain and disordered loop of the GAF domain [35]. It was shown for iRFP713 and IFP1.4 that the cleavage sites can be introduced mainly in the N terminal disordered region of the PAS domain, disordered loop between the PAS and GAF domains, GAF domain α helix adjacent to this loop, and in the loops between the β strands in the PAS domain [51,52]. NIR FPs do not tolerate circular permutations between the sites in the GAF domain loop participating in the knot formation [52].…”

“…It was shown for iRFP713 and IFP1.4 that the cleavage sites can be introduced mainly in the N terminal disordered region of the PAS domain, disordered loop between the PAS and GAF domains, GAF domain α helix adjacent to this loop, and in the loops between the β strands in the PAS domain [51,52]. NIR FPs do not tolerate circular permutations between the sites in the GAF domain loop participating in the knot formation [52]. These data argue for that some PPI reporters based on iRFPs and IFPs may fold into the knotted structure (typical for the native pro tein) at reconstruction from the two protein fragments.…”

Circularly Permuted Far-Red Fluorescent Proteins

“…Instead, they have to bind a biliverdin cofactor for fluorescence. A recent study performed circular permutation on iRFP, a biliverdin-based NIFP, and discovered several variants exhibiting near-infrared (NIR) fluorescence in E. coli and cultured HeLa cells [ 19 ]. In other studies, NIR-GECO1 and NIR-GECO2, two GEFPIs for Ca 2+ , were derived by inserting calcium-binding elements into biliverdin-binding mIFP [ 20 , 21 ].…”