Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.
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 iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs 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 that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs 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 quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal 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 toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.
Department of Biosciences, Rice University , Houston, Texas 77005, United States.|Biochemistry and Cell Biology Graduate Program, Rice University , Houston, Texas 77005, United States.
Naresh
N
Pandey
Systems, Synthetic, and Physical Biology Graduate Program, Rice University , Houston, Texas 77005, United States.|Department of Chemical and Biomolecular Engineering, Rice University , Houston, Texas 77005, United States.
Brianna E
BE
Kuypers
Department of Biosciences, Rice University , Houston, Texas 77005, United States.
Barbara
B
Nassif
Department of Biosciences, Rice University , Houston, Texas 77005, United States.|Biochemistry and Cell Biology Graduate Program, Rice University , Houston, Texas 77005, United States.
Emily E
EE
Thomas
Department of Biosciences, Rice University , Houston, Texas 77005, United States.|Biochemistry and Cell Biology Graduate Program, Rice University , Houston, Texas 77005, United States.
Razan N
RN
Alnahhas
Department of Biosciences, Rice University , Houston, Texas 77005, United States.|Department of Chemical and Biomolecular Engineering, Rice University , Houston, Texas 77005, United States.|Department of Bioengineering, Rice University , Houston, Texas 77005, United States.
Laura
L
Segatori
Department of Biosciences, Rice University , Houston, Texas 77005, United States.|Department of Bioengineering, Rice University , Houston, Texas 77005, United States.
Jonathan J
JJ
Silberg
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D004926:Escherichia coli / Q000378:metabolism*; D005434:Flow Cytometry; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry*; D010834:Phytochrome / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D019265:Spectroscopy, Near-Infrared
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D010834:Phytochrome