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Long-lived bright red emitting azaoxa-triangulenium fluorophores.

Maliwal BP, Fudala R, Raut S, Kokate R, Sørensen TJ, Laursen BW, Gryczynski Z, Gryczynski I - PLoS ONE (2013)

Bottom Line: Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns.Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States of America. bpmal001@gmail.com

ABSTRACT
The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

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Related in: MedlinePlus

Fluorescence anisotropy decays of labeled proteins.(A) ADOTA Avidin and ADOTA-IgG (B) DAOTA-Streptavidin and DAOTA-IgG.
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pone-0063043-g009: Fluorescence anisotropy decays of labeled proteins.(A) ADOTA Avidin and ADOTA-IgG (B) DAOTA-Streptavidin and DAOTA-IgG.

Mentions: The results from analysis of anisotropy decays of labeled proteins are given in Table 4. The anisotropy decays are shown in Figure 9A and 9B for ADOTA labeled IgG and avidin and DAOTA labeled streptavidin and IgG, respectively. The anisotropy decays are heterogeneous, with presence of up to three correlation times. The sub-nanosecond to nanosecond correlation time represents local motional freedom of the labeled probe. Somewhat slower intermediate motions of 3 ns to 6 ns correlation times are likely due to segmental motions of secondary structure elements. The longest correlation times reflect over all tumbling of the protein macromolecules. The correlation times of 80 ns to 85 ns in case of IgG (MW 150 kDa to 160 kDa), 25 ns in case of streptavidin (MW 54 kDa) and 37 ns for avidin (MW 64 kDa) are reasonable for respective asymmetrical globular proteins. The anisotropy fraction associated with rotational diffusion of proteins in case of DAOTA is between 0.129 and 0.158 when compared to the value of 0.066 to 0.076 with ADOTA. This may reflect more hydrophobic character of DAOTA and enhanced propensity to stick to protein surface when compared with ADOTA.


Long-lived bright red emitting azaoxa-triangulenium fluorophores.

Maliwal BP, Fudala R, Raut S, Kokate R, Sørensen TJ, Laursen BW, Gryczynski Z, Gryczynski I - PLoS ONE (2013)

Fluorescence anisotropy decays of labeled proteins.(A) ADOTA Avidin and ADOTA-IgG (B) DAOTA-Streptavidin and DAOTA-IgG.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646960&req=5

pone-0063043-g009: Fluorescence anisotropy decays of labeled proteins.(A) ADOTA Avidin and ADOTA-IgG (B) DAOTA-Streptavidin and DAOTA-IgG.
Mentions: The results from analysis of anisotropy decays of labeled proteins are given in Table 4. The anisotropy decays are shown in Figure 9A and 9B for ADOTA labeled IgG and avidin and DAOTA labeled streptavidin and IgG, respectively. The anisotropy decays are heterogeneous, with presence of up to three correlation times. The sub-nanosecond to nanosecond correlation time represents local motional freedom of the labeled probe. Somewhat slower intermediate motions of 3 ns to 6 ns correlation times are likely due to segmental motions of secondary structure elements. The longest correlation times reflect over all tumbling of the protein macromolecules. The correlation times of 80 ns to 85 ns in case of IgG (MW 150 kDa to 160 kDa), 25 ns in case of streptavidin (MW 54 kDa) and 37 ns for avidin (MW 64 kDa) are reasonable for respective asymmetrical globular proteins. The anisotropy fraction associated with rotational diffusion of proteins in case of DAOTA is between 0.129 and 0.158 when compared to the value of 0.066 to 0.076 with ADOTA. This may reflect more hydrophobic character of DAOTA and enhanced propensity to stick to protein surface when compared with ADOTA.

Bottom Line: Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns.Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States of America. bpmal001@gmail.com

ABSTRACT
The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

Show MeSH
Related in: MedlinePlus