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)|
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Jul 25 2006|
- Biliprotein photoreceptor
- Single-molecule fluorescence
ASJC Scopus subject areas