Abstract
Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1Δ, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1Δ apoprotein (apoCph1Δ) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1Δ altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1Δ and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 11136-11141 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 103 |
| Issue number | 30 |
| DOIs | |
| State | Published - Jul 25 2006 |
| Externally published | Yes |
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Keywords
- Biliprotein photoreceptor
- Biophotonics
- Phytofluor
- Single-molecule fluorescence
ASJC Scopus subject areas
- Genetics
- General
Cite this
Single-molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy. / Miller, Abigail E.; Fischer, Amanda J.; Laurence, Ted; Hollars, Christopher W.; Saykally, Richard J.; Lagarias, J. Clark; Huser, Thomas R.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 30, 25.07.2006, p. 11136-11141.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Single-molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy
AU - Miller, Abigail E.
AU - Fischer, Amanda J.
AU - Laurence, Ted
AU - Hollars, Christopher W.
AU - Saykally, Richard J.
AU - Lagarias, J. Clark
AU - Huser, Thomas R
PY - 2006/7/25
Y1 - 2006/7/25
N2 - Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1Δ, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1Δ apoprotein (apoCph1Δ) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1Δ altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1Δ and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein.
AB - Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1Δ, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1Δ apoprotein (apoCph1Δ) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1Δ altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1Δ and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein.
KW - Biliprotein photoreceptor
KW - Biophotonics
KW - Phytofluor
KW - Single-molecule fluorescence
UR - http://www.scopus.com/inward/record.url?scp=33746612757&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33746612757&partnerID=8YFLogxK
U2 - 10.1073/pnas.0604724103
DO - 10.1073/pnas.0604724103
M3 - Article
C2 - 16844775
AN - SCOPUS:33746612757
VL - 103
SP - 11136
EP - 11141
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 30
ER -