Cyanochrome fluorophores
Genetically-engineered cyanochrome fluorophore molecules (fluorophores) with increased fluorescence and with absorbing fluorescence in the blue and green (blue/green) portion of the light spectrum are provided. These fluorophores are derived from the domains of phytochromes, and in particular cyanobacterial phytochromes. Methods for generating these fluorophores and various applications of these fluorophores are also provided.
1. An isolated polypeptide comprising a modified blue/green light absorbing Thermosynechococcus elongatus phytochrome domain, wherein the modified phytochrome domain has at least 85% amino acid sequence identity to the amino acid sequence of positions 1 to 162 of SEQ ID NO: 1, wherein the modified phytochrome domain has an amino acid other than Asp at the residue corresponding to position 63 of SEQ ID NO: 1 or an amino acid other than Asp at the residue corresponding to position 63 of SEQ ID NO: 1 in combination with an amino acid other than Cys at the residue corresponding to position 65 of SEQ ID NO: 1, and wherein the modified phytochrome domain has increased fluorescent activity compared with fluorescent activity of a phytochrome domain comprising the amino acid sequence from positions 1 to 162 of SEQ ID NO: 1.
2. The isolated polypeptide of claim 1 , wherein the modified phytochrome domain has at least 95% amino acid sequence identity to the amino acid sequence of positions 1 to 162 of SEQ ID NO: 1.
3. The isolated polypeptide of claim 1 , wherein the modified phytochrome domain has an amino acid other than Asp at the residue corresponding to position 63 of SEQ ID NO: 1, and wherein the amino acid other than Asp is selected from the group consisting of His, Asn, and Ala.
4. The isolated polypeptide of claim 1 , wherein the modified phytochrome domain has an amino acid other than Cys at the residue corresponding to position 126 of SEQ ID NO: 1.
5. A method of determining whether a first test polypeptide interacts with a second test polypeptide, the method comprising
exposing a first test polypeptide comprising the isolated polypeptide of claim 1 to a second test polypeptide linked to a chromophore, and
measuring the presence or absence of fluorescence emitted from the chromophore,
wherein the presence of fluorescence emitted from the chromophore indicates the first test polypeptide interacts with the second test polypeptide.
6. The method of claim 5 , wherein the chromophore comprises a modified Thermosynechococcus elongatus phytochrome domain having the amino acid sequence of positions 1 to 162 of SEQ ID NO: 1, except Cys at the residue corresponding to position 65 of SEQ ID NO: 1 is replaced with a different amino acid.
7. A method for localizing a polypeptide of interest in an isolated cell comprising
providing an isolated cell comprising a fusion protein, wherein the fusion protein comprises the polypeptide of interest linked to the isolated polypeptide of claim 1 , and
detecting the location in the isolated cell of the fusion protein.
8. An isolated modified blue/green light absorbing phytochrome domain, wherein the modified phytochrome domain has at least 85% amino acid sequence identity to the amino acid sequence of positions 1 to 162 of SEQ ID NO: 1, wherein the modified phytochrome domain has an amino acid other than Asp at the residue corresponding to position 63 of SEQ ID NO: 1, and wherein the phytochrome domain has increased fluorescent activity compared with fluorescent activity of a phytochrome domain comprising the amino acid sequence from positions 1 to 162 of SEQ ID NO: 1.
9. The isolated modified blue/green light absorbing phytochrome domain of claim 8 , wherein the modified phytochrome domain has an amino acid other than Asp at the residue corresponding to position 63 of SEQ ID NO: 1, and wherein the amino acid other than Asp is selected from the group consisting of His, Asn, and Ala.
10. The isolated polypeptide of claim 1 , wherein the amino acid other than Cys at the residue corresponding to position 65 of SEQ ID NO: 1 is His or Ala.
11. The isolated polypeptide of claim 4 , wherein the amino acid other than Cys at the residue corresponding to position 126 of SEQ ID NO: 1 is Ala.