Picosecond Time-Resolved FRET in the Fluorescent Protein fromDiscosoma Red (wt-DsRed)

Abstract

Ultrafast, intra‐oligomer fluorescence resonance energy transfer (FRET) between an immature green‐emitting GFP‐like chromophore to the mature red‐emitting chromophore is found in the novel red fluorescent protein wt‐DsRed (the picture shows the steady‐state absorption (solid line) and emission (dotted) spectra). Since FRET is by its very nature a short range process, it represents a highly suitable method to probe oligomerization. This work describes a method preferentially applicable to the efficient screening of protein variants with mutagenetically altered surface docking sites.

New Insights into the Photophysics of DsRed by Multiparameter Spectroscopy on Single Proteins

“…The spread of the decay constants agrees well with the spread of lifetimes of single green fluorescent protein variants reported recently . The most likely lifetimes are in good agreement with numbers reported for bulk measurements in literature , and our own ensemble results: Ensemble lifetime measurements with a 590 nm bandpassfilter in front of the detection unit and a excitation wavelength λ exc = 561 nm revealed a monoexponential decay curve with a lifetime of τ = 3.7 ns. Hence we assign the long-lived component of our single tetramer measurement to the red chromophore.…”

“…Time resolved measurements of this subpopulation on the single tetramer level revealed biexponential decay curves with time constants in the range of τ 1 = 0.3−0.5 ns and τ 2 = 1.6−2.2 ns. The shorter lived component fits well to the numbers reported in literature. , However, we do not attribute this component to an unquenched donor emission but rather to an energy transfer from the green emitting to the yellow from. Note that this yellow form can only be accounted for in fluorescence decay curves when no spectral filtering is used.…”

“…Figure a depicts the fluorescence decay of a DsRed ensemble upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the red (λ det = 600 nm). We observe comparable time constants (τ 1 = 1.8 ns (5%) and τ 2 = 3.7 ns (95%)) as reported in literature. , …”

“…Upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the green (λ det = 505 nm) we obtained a fluorescence intensity decay which can be well fitted to a three-exponential decay function as shown in Figure b. Here we found two short-lived components of τ 1 = 0.12 ns (89%) and τ 2 = 1.1 ns (8%) which agree well with decay constants reported in literature. , However, we observed an additional long-lived component with a decay constant of τ 3 = 5.1 ns with a contribution of about 3% to the total decay. The assignment of the decay times obtained from ensemble measurements to distinct relaxation processes within in the DsRed tetramer is exceedingly difficult due to the highly complex photophysics of the DsRed system.…”

New Insights into the Photophysics of DsRed by Multiparameter Spectroscopy on Single Proteins

“…The spread of the decay constants agrees well with the spread of lifetimes of single green fluorescent protein variants reported recently . The most likely lifetimes are in good agreement with numbers reported for bulk measurements in literature , and our own ensemble results: Ensemble lifetime measurements with a 590 nm bandpassfilter in front of the detection unit and a excitation wavelength λ exc = 561 nm revealed a monoexponential decay curve with a lifetime of τ = 3.7 ns. Hence we assign the long-lived component of our single tetramer measurement to the red chromophore.…”

“…Time resolved measurements of this subpopulation on the single tetramer level revealed biexponential decay curves with time constants in the range of τ 1 = 0.3−0.5 ns and τ 2 = 1.6−2.2 ns. The shorter lived component fits well to the numbers reported in literature. , However, we do not attribute this component to an unquenched donor emission but rather to an energy transfer from the green emitting to the yellow from. Note that this yellow form can only be accounted for in fluorescence decay curves when no spectral filtering is used.…”

“…Figure a depicts the fluorescence decay of a DsRed ensemble upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the red (λ det = 600 nm). We observe comparable time constants (τ 1 = 1.8 ns (5%) and τ 2 = 3.7 ns (95%)) as reported in literature. , …”

“…Upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the green (λ det = 505 nm) we obtained a fluorescence intensity decay which can be well fitted to a three-exponential decay function as shown in Figure b. Here we found two short-lived components of τ 1 = 0.12 ns (89%) and τ 2 = 1.1 ns (8%) which agree well with decay constants reported in literature. , However, we observed an additional long-lived component with a decay constant of τ 3 = 5.1 ns with a contribution of about 3% to the total decay. The assignment of the decay times obtained from ensemble measurements to distinct relaxation processes within in the DsRed tetramer is exceedingly difficult due to the highly complex photophysics of the DsRed system.…”

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals ¶†

“…We note that our simulation involved use of distributed kinetic parameters, whereas all others used discretized exponential components, which may lead to apparent kinetic discrepancies. Nonetheless, given the similar time constants, we suggest in vitro estimates of center‐to‐center intermolecular distances between FP (41), which are consistent with X‐ray crystallographic data (43), may also be qualitatively comparable with our in vivo data. Although, as mentioned above, we have no direct way to measure the absolute extinctions or molecular orientations in vivo.…”

New Insights into the Photophysics of DsRed by Multiparameter Spectroscopy on Single Proteins

“…The spread of the decay constants agrees well with the spread of lifetimes of single green fluorescent protein variants reported recently . The most likely lifetimes are in good agreement with numbers reported for bulk measurements in literature , and our own ensemble results: Ensemble lifetime measurements with a 590 nm bandpassfilter in front of the detection unit and a excitation wavelength λ exc = 561 nm revealed a monoexponential decay curve with a lifetime of τ = 3.7 ns. Hence we assign the long-lived component of our single tetramer measurement to the red chromophore.…”

“…Time resolved measurements of this subpopulation on the single tetramer level revealed biexponential decay curves with time constants in the range of τ 1 = 0.3−0.5 ns and τ 2 = 1.6−2.2 ns. The shorter lived component fits well to the numbers reported in literature. , However, we do not attribute this component to an unquenched donor emission but rather to an energy transfer from the green emitting to the yellow from. Note that this yellow form can only be accounted for in fluorescence decay curves when no spectral filtering is used.…”

“…Figure a depicts the fluorescence decay of a DsRed ensemble upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the red (λ det = 600 nm). We observe comparable time constants (τ 1 = 1.8 ns (5%) and τ 2 = 3.7 ns (95%)) as reported in literature. , …”

“…Upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the green (λ det = 505 nm) we obtained a fluorescence intensity decay which can be well fitted to a three-exponential decay function as shown in Figure b. Here we found two short-lived components of τ 1 = 0.12 ns (89%) and τ 2 = 1.1 ns (8%) which agree well with decay constants reported in literature. , However, we observed an additional long-lived component with a decay constant of τ 3 = 5.1 ns with a contribution of about 3% to the total decay. The assignment of the decay times obtained from ensemble measurements to distinct relaxation processes within in the DsRed tetramer is exceedingly difficult due to the highly complex photophysics of the DsRed system.…”

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals ¶†

“…We note that our simulation involved use of distributed kinetic parameters, whereas all others used discretized exponential components, which may lead to apparent kinetic discrepancies. Nonetheless, given the similar time constants, we suggest in vitro estimates of center‐to‐center intermolecular distances between FP (41), which are consistent with X‐ray crystallographic data (43), may also be qualitatively comparable with our in vivo data. Although, as mentioned above, we have no direct way to measure the absolute extinctions or molecular orientations in vivo.…”

New Insights into the Photophysics of DsRed by Multiparameter Spectroscopy on Single Proteins

“…The spread of the decay constants agrees well with the spread of lifetimes of single green fluorescent protein variants reported recently . The most likely lifetimes are in good agreement with numbers reported for bulk measurements in literature , and our own ensemble results: Ensemble lifetime measurements with a 590 nm bandpassfilter in front of the detection unit and a excitation wavelength λ exc = 561 nm revealed a monoexponential decay curve with a lifetime of τ = 3.7 ns. Hence we assign the long-lived component of our single tetramer measurement to the red chromophore.…”

“…Time resolved measurements of this subpopulation on the single tetramer level revealed biexponential decay curves with time constants in the range of τ 1 = 0.3−0.5 ns and τ 2 = 1.6−2.2 ns. The shorter lived component fits well to the numbers reported in literature. , However, we do not attribute this component to an unquenched donor emission but rather to an energy transfer from the green emitting to the yellow from. Note that this yellow form can only be accounted for in fluorescence decay curves when no spectral filtering is used.…”

“…Figure a depicts the fluorescence decay of a DsRed ensemble upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the red (λ det = 600 nm). We observe comparable time constants (τ 1 = 1.8 ns (5%) and τ 2 = 3.7 ns (95%)) as reported in literature. , …”

“…Upon excitation in the blue spectral region (λ exc = 467 nm) and detection in the green (λ det = 505 nm) we obtained a fluorescence intensity decay which can be well fitted to a three-exponential decay function as shown in Figure b. Here we found two short-lived components of τ 1 = 0.12 ns (89%) and τ 2 = 1.1 ns (8%) which agree well with decay constants reported in literature. , However, we observed an additional long-lived component with a decay constant of τ 3 = 5.1 ns with a contribution of about 3% to the total decay. The assignment of the decay times obtained from ensemble measurements to distinct relaxation processes within in the DsRed tetramer is exceedingly difficult due to the highly complex photophysics of the DsRed system.…”

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals ¶†

“…We note that our simulation involved use of distributed kinetic parameters, whereas all others used discretized exponential components, which may lead to apparent kinetic discrepancies. Nonetheless, given the similar time constants, we suggest in vitro estimates of center‐to‐center intermolecular distances between FP (41), which are consistent with X‐ray crystallographic data (43), may also be qualitatively comparable with our in vivo data. Although, as mentioned above, we have no direct way to measure the absolute extinctions or molecular orientations in vivo.…”