2.1.

Clone Construction

The construction of Venus C1 (containing the A206K monomeric mutation), Cerulean C1 (containing the A206K monomeric mutation), 6xHis tagged Cerulean, 6xHis tagged Venus, Cerulean-5-amber (C5A), Venus-5-amber (V5A), Venus-5-Venus (V5V), Cerulean-Traf-Venus (CTV), Cerulean-K-Ras (C-K-Ras) are described elsewhere.^{10, 20} The open reading frame (ORF) of Cerulean without the translation initiation codon (ATG) was amplified by PCR using pfu ultra polymerase (Stratgene). A BglII site (underlined) and an EcoRI site were engineered into the sense $5^{\prime }$ -GCAGATCTGTGAGCAAGGGCGAGGAGCTGTTCACC- $3^{\prime }$ and antisense $5^{\prime }$ -GCGAATTCCTTGTACAGCTCGTCCATGCCGAGAGTG- $3^{\prime }$ primers, respectively, for directional cloning into the Cerulean C1 vector. The PCR fragment was cloned into the Zero Blunt-II Topo vector (Invitrogen) and sequenced. The sequence-verified cDNA for Cerulean was subsequently cloned into the BglII/EcoRI digested Cerulean C1 to generate C5C. The ORF of Cerulean without the first two codons (ATG GTG) was amplified using oligos containing an Asp718 (Roche) site on the sense primer (underlined) $5^{\prime }$ -AGTCGGTACCAGCAAGGGCGAGGAGCTGTT- $3^{\prime }$ and a BamHI (New England Biolabs) site on the antisense primer (underlined) $5^{\prime }$ -AGTCTCGGATCCCTTGTACAGCTCGTCCATGCCGAGAGTGATC-3. The ORF was cloned into Asp718/BamHI (New England Biolabs) digested Cerulean C1 to generate C17C. The C32C clone was generated by excising the Cerulean ORF from C32V (Ref. 20) using Nhel/Agel (New England Biolabs) and cloning it into Cerulean N1. The cDNA coding for Traf was excised from CTV using BspE1 and cloned into C5A or V5A to generate CTA and VTA, respectively. The oligonucleotide coding for the K-Ras²¹ membrane targeting sequence was excised from Cerulean-K-Ras²⁰using BsrG1 and NotI (New England Biolabs). The insert was cloned into linearized Venus N1 and EGFP N1 to generate V-K-Ras and EGFP-K-Ras, respectively.

2.2.

Protein Purification

BL21(DE3)pLysS bacteria (Invitrogen) transformed with either His tagged Cerulean or Venus were plated onto LB Agar plates with chloramphenicol and ampicillin and grown overnight at $37°\mathrm{C}$ . Five ml of liquid LB cultures with chloramphenicol and ampicillin were inoculated with a single colony of either Cerulean or Venus expressing bacteria and shaken overnight at $37°\mathrm{C}$ . The next day, a $1\text{-}\mathrm{ml}$ bacterial culture was added to $100\mathrm{ml}$ fresh LB with antibiotics and the culture flasks were shaken until the ${\mathrm{OD}}_{600}$ of the culture reached 0.4 to 0.6. Protein expression was induced by adding $1\mathrm{ml}$ of 1M IPTG (Sigma), and cell cultures were incubated at room temperature overnight, then centrifuged and frozen $(-80°\mathrm{C})$ . Before purification, the cells were thawed, resuspended in lysis buffer ( $50\mathrm{mM}$ ${\mathrm{NaH}}_2{\mathrm{PO}}_4$ , $300\mathrm{mM}$ $\mathrm{NaCl}$ , $20\mathrm{mM}$ Imidazole), and sonicated. Protein was purified by $\mathrm{Ni}$ -NTA magnetic agarose beads (Qiagen) using manufacturer suggestions.

2.3.

Refractive Index $\left(\eta \right)$ Studies

FP dilutions containing 0, 10, 30, 50, and 70% glycerol (w/w) (MG Scientific Inc.) with identical concentrations of either Cerulean or Venus ( $5\mathrm{\mu }\mathrm{M}$ each) were prepared. The refractive index of each dilution was measured at room temperature using a refractometer (Milton Roy Company). The dilutions were loaded into $0.1\text{-}\mathrm{mm}\text{-}\mathrm{square}$ capillaries (Vitrocon), and the FP lifetimes were measured by time-correlated single-photon counting (TCSPC).²²

2.4.

Microscopy and Imaging

All imaging and spectroscopy was performed using a Zeiss 510 META/NLO scan head attached to an upright Zeiss Axioplan 2 microscope. A mode-locked Ti:sapphire laser (Coherent Chameleon), tunable from 710 to $950\mathrm{nm}$ , was attached to the NLO laser “in” port of the scan head through an acoustic optic modulator.

2.5.

Spectral Imaging

Spectral images of Cerulean-K-Ras, EGFP-K-Ras, and Venus-K-Ras were acquired using a $40\times 0.8$ numerical aperture water immersion objective with the Ti:sapphire laser mode-locked at $880\mathrm{nm}$ , as previously described in Ref. 23.

2.6.

FLIM-FRET Measurements

Time-domain FLIM and spectroscopy were performed using TCSPC.²² Cerulean constructs were excited with a mode-locked laser tuned to $820\mathrm{nm}$ . GFP constructs were excited with $850\text{-}\mathrm{nm}$ excitation. Venus constructs were excited with $950\text{-}\mathrm{nm}$ excitation. For lifetime determinations, emitted photons were filtered through a BG39 filter, a polarizer set to $54.7\mathrm{deg}$ , a $700\text{-}\mathrm{nm}$ short-pass filter (Chroma Optical, e700sp-2p), and either a 460- to $490\text{-}\mathrm{nm}$ bandpass filter for Cerulean or a 520- to $550\text{-}\mathrm{nm}$ filter for Venus. They were detected on a bialkali microchannel plate photomultiplier (Hamamatsu R3809U-52) attached to a Zeiss 510 non-descanned detector port placed in the transmitted light pathway. Photons emitted from all the pixels of a FLIM image were pooled, correlated with excitation laser pulses using a Becker and Hickl SPC830 module to generate a lifetime spectrum. The fluorescence lifetimes $\left(\tau \right)$ of the constructs were determined using SPCImage software using a measured system response.²⁰ The FRET efficiency $\left(E\right)$ of the constructs were calculated from lifetimes using Eq. 1:

2.7.

Time-Dependent Anisotropy Measurements

For time-dependent fluorescence anisotropy measurements, a mode-locked Ti:sapphire laser was scanned over a region of interest and detected photons pooled, counted, and correlated with excitation laser pulses to generate a fluorescence lifetime decay curve. The polarizer was set first at $0\mathrm{deg}$ and then repeated with the polarizer set at $90\mathrm{deg}$ relative to the laser polarization. The decay curves obtained from parallel and perpendicular polarization settings were exported into Igor Pro 5.04 (Wavemetrics) and the fluorescence anisotropy $\left(r\right)$ was calculated using the following equation:

2.8.

Simultaneous Anisotropy and Lifetime Measurements

For simultaneous acquisition of anisotropy and lifetime curves, a Ti:sapphire laser mode-locked at $820\mathrm{nm}$ was scanned over a region of interest. The emitted photons were filtered through a BG39 filter and a polarizing beamsplitter that separated the photons parallel and perpendicular to the excitation light source. The parallel and perpendicular photons were counted using two bialkali microchannel plate photomultipliers (Hamamatsu R3809U-52) attached to a Zeiss 510 non-descanned detector port placed in the transmitted light pathway. Anisotropy curves for each clone (at least six cells each) were generated using Eq. 2 as described earlier. Since the parallel and perpendicular data were collected through different optical paths and different detectors, the $G$ factor was found to be 1.26.

Fluorescence lifetime curves were generated using the equation

3.1.

EM-RET affects the lifetime of Cerulean

Since the fluorescence lifetime of a fluorophore is typically unaffected by the intensity of incident light or the concentration of fluorophores, FLIM is frequently used to calculate hetero-FRET efficiencies in living cells. The fluorescence lifetime $\left(\tau \right)$ of a molecule is a measure of the average time a fluorophore spends in the excited state.⁶ FRET provides a competing pathway by which excited state energy can decay, so FRET results in a faster decay time. Unfortunately, any additional mechanism that decreases the donor fluorescence lifetime such as photobleaching, dynamic quenching, or changes in refractive index $\left(\eta \right)$ might be misinterpreted as FRET. To test if the lifetime of the Cerulean was altered in the presence of EM-RET, a DNA construct called C5C encoding two Ceruleans separated by a 5 amino acid linker was generated. Three additional related constructs, composed of Cerulean, Venus,²⁴ or Amber (a point mutation in Venus that destroys the fluorophore²⁰) called C5A, V5A, and V5V, were also generated as controls for C5C. In all live-cell experiments, the Cerulean intensity was monitored throughout the data acquisition period to ensure that the fluorophore did not bleach, because even small amounts of photobleaching can alter the lifetime.²⁵ Fluorescence anisotropy curves of Cerulean, C5A, and C5C are shown in Fig. 1a . The anisotropy decay of cells transfected with Cerulean (green circles) or C5A (red inverted triangles) were both well fit by a single exponential with time constants $\left(\varphi \right)$ of $16.7\pm 1.1\mathrm{ns}$ ( $\mathrm{mean}\pm \mathrm{SD}$ $n=5$ ) and $25.0\pm 1.8\mathrm{ns}$ $(n=5)$ , respectively. These time constants correspond to the molecular rotation of the Cerulean monomer and the Cerulean in C5A construct. Note that the Cerulean in C5A rotated slightly slower than Cerulean alone, as expected for a larger construct. In contrast, C5C has two functional fluorophores in close proximity, so energy migration (EM-RET) can occur between the two Cerulean subunits. As shown in Fig. 1a, the anisotropy decay curve obtained from cells transfected with C5C had two decay components (blue triangles). C5C had a slow rotational decay component similar to C5A and a fast component (observed between 0 to $3\mathrm{ns}$ ) corresponding to EM-FRET.

Fig. 1

Attenuation of Cerulean’s fluorescence lifetime correlates with EM-RET. (a) C5C exhibits EM-RET (blue triangles) as seen in the rapid depolarization detected in the early part of the anisotropy decay (single arrow) when compared to C5A (red inverted triangles) and Cerulean (green filled circles). Also note that the rotational component of the anisotropy curves (double arrows) of C5A and C5C are slower than Cerulean due to increased mass. Furthermore, because all experiments were conducted using two-photon excitation, our $r_0>0.40$ . (b) Anisotropy curve of V5V, like that of C5C, also exhibits EM-RET. (c) Fluorescence lifetimes of Cerulean, C5A, and C5C (bluebars), C5V (red bar) data orginally used to determine FRET efficiency of C5V in (Ref. 20) and Venus, V5A and V5V (yellow-green bars). C5A and C5C have significantly different fluorescence lifetimes compared to Cerulean alone. Similarly, V5A and V5V have significantly different lifetimes than Venus alone, suggesting that both Cerulean and Venus lifetimes are affected by attaching a protein to them. The fluorescence lifetime of C5C is significantly different from C5A, while fluorescence lifetimes of V5V and V5A are not different. (Color online only.)

The fluorescent lifetimes $\left(\tau \right)$ of Cerulean, C5A, and C5C were measured using TCSPC of cells transfected with these constructs [Fig. 1c]. The fluorescence lifetimes were calculated for individual curves using SPCImage and could be fit to a single exponential based on the ${\chi }^2$ rule.²⁰ Other laboratories have shown that Cerulean decays as a double exponential.²⁶ Cerulean had a $\tau$ of $2.99\pm 0.11\mathrm{ns}$ $(\mathrm{mean}\pm \mathrm{SD},n=21)$ , C5A decayed slightly faster with a $\tau$ of $2.91\pm 0.05\mathrm{ns}$ $(n=21)$ , and C5C decayed the fastest with a $\tau$ of $2.78\pm 0.62\mathrm{ns}$ $(n=21)$ . These lifetimes were all significantly different from each other as determined by an analysis of variance (ANOVA). Apparently, the simple act of attaching a protein to Cerulean can slightly alter its fluorescence lifetime decay, as seen in the difference in $\tau$ between C5A and Cerulean. Furthermore, because the $\tau$ of C5C was significantly faster than that of C5A (Tukey’s multiple comparison test $p<0.001$ ), energy migration might be responsible for the further decrease in the Cerulean’s fluorescence lifetime. The ${\tau }_{\mathrm{ave}}$ of C5V fit to a double exponential (based on the ${\chi }^2$ rule) from previous experiments was included [Fig. 1c red bar] to assist in visualizing the magnitude of lifetime attenuation seen in Cerulean.²⁰

To test if an energy migration correlated reduction in fluorescent lifetime is a unique attribute shared by CFP and Cerulean as opposed to a general characteristic of spectral variants of GFP, we measured the fluorescence anisotropy decay [Fig. 1b] and fluorescence lifetime decay [Fig. 1c] of Venus, and the related yellow fluorescent constructs V5A and V5V. The anisotropy decay of Venus was virtually indistinguishable from the decay of Cerulean with a $\varphi$ of $17.0\pm 1.4\mathrm{ns}$ ( $\mathrm{mean}\pm \mathrm{SD}$ , $n=6$ ). Similarly, the anisotropy decay of V5A was virtually indistinguishable from the decay of C5A with a $\varphi$ of $22.7\pm 1.6\mathrm{ns}$ $(n=4)$ . Like C5C, V5V was well fit by a double exponential, indicating the presence of energy migration in this construct. The amplitude of V5V’s fast decay component was larger than that of C5C [compare bottom trace in Figs. 1a and 1b]. This is indicative of a faster energy transfer rate in V5V than in C5C and is consistent with the measured Förster distance for Cerulean-Cerulean RET $\left(3.52\mathrm{nm}\right)$ and Venus-Venus RET $\left(4.95\mathrm{nm}\right)$ .²⁷ An ANOVA revealed that the fluorescence lifetime for the Venus monomer ( $3.25\pm 0.10\mathrm{ns}$ , $n=20$ ) [Fig. 1c] was significantly different from the lifetimes for V5A ( $3.10\pm 0.05\mathrm{ns}$ , $n=20$ ) and V5V ( $3.12\pm 0.08\mathrm{ns}$ , $n=21$ ). (Tukey’s multiple comparison test $p<0.001$ ). These results demonstrate that the addition of protein adducts to either Cerulean or Venus can result in a slightly faster fluorescence lifetime decay. Additionally, since the fluorescence lifetimes of V5A and V5V were indistinguishable from each other (Tukey’s multiple comparison test $p>0.05$ ), the Venus constructs did not demonstrate a further reduction in lifetimes that were correlated with energy migration.

3.2.

Refractive index affects fluorescence lifetimes

We were interested in understanding why attaching a protein to either Cerulean or Venus resulted in a slight yet significant reduction in fluorescence lifetime. It is known that a change in refractive index alters the fluorescence lifetimes of FPs. Refractive indices for proteins ( $\eta =1.58$ to 1.613) to (Ref. 28) and lipids ( $\eta =1.4$ to 1.6) (Ref. 29) are larger than that of cytosol $(\eta =1.38)$ (Ref. 30). Therefore, adding a protein to a fluorophore or targeting a fluorophore to a lipid environment might decrease its lifetime.^{29, 31, 32} The relationship between refractive indices and lifetimes can be seen for purified Cerulean and Venus (Fig. 2 ). Cerulean or Venus was diluted into different concentrations of glycerol, and the refractive index $\left(\eta \right)$ and $\tau$ for each sample were measured. As predicted by the Strickler-Berg equation,³³ $\tau$ got shorter as the refractive index increased. This supports the idea that attaching a protein adduct to a FP may alter the refractive index that its fluorophore perceives, thus altering its lifetime.

Fig. 2

Refractive index $\left(\eta \right)$ dependence of FP lifetimes. Cerulean and Venus fluorescence lifetimes decrease with increasing refractive index. Changes in the lifetime of Venus were less than that observed in Cerulean. Note the lifetime of a FP attached to another protein or membrane is predicted to be faster than that of an FP in cytosol as a result of being in an environment with a higher $\eta$ .

We have shown that a construct containing two covalently linked Cerulean fluorophores has a shorter $\tau$ than a Cerulean covalently attached to Amber. This was not observed for a nearly identical construct comprised of two covalently linked Venus molecules (Fig. 1). This anomalous behavior appears to be correlated with energy migration between Cerulean molecules. We next wanted to test if a covalent attachment between two Ceruleans was required for this attenuation of lifetime. DNA constructs encoding either Cerulean, Venus, or EGFP ligated to the membrane-targeting domain of the monomeric GTP binding protein K-Ras were produced and transfected into HEK 293 cells. A typical plasma membrane localization was observed for these constructs [Figs. 3a, 3b, 3c ]. Unlike cytoplasmic proteins, overexpressed membrane proteins are trapped in the same small compartment and thus readily reach local concentrations sufficient to support energy migration.³⁴ All K-Ras constructs, when examined by anisotropy decay analysis, exhibited EM-RET (data not shown). In contrast, only Cerulean-K-Ras had a fluorescence lifetime that was significantly shorter than the lifetime of the FP alone [Fig. 3d], suggesting that the covalent attachment between Ceruleans is not required for this effect. The lifetimes of Venus and Venus-K-Ras were indistinguishable, as were the lifetimes of EGFP and EGFP-K-Ras (unpaired t-test, $p>0.05$ ). The lack of a decrease in the lifetimes of Venus or EGFP, even though they were targeted to the plasma membrane (which has a high $\eta$ ), suggests that refractive index changes may not completely account for the lifetime changes seen in the V5A and C5A constructs. This might be explained by the fact that FPs respond to refractive index changes over a distance range of micrometers.^{28, 31} An alternative explanation for the shortening of the C5A and V5A lifetimes could be due to the addition of a bulky protein that may alter the shape of their $\beta$ -barrels. The absence of fluorescence lifetime attenuation with the K-Ras membrane targeting domain,²¹ a small 22 amino acid polypeptide added to the C-terminus of either Venus or GFP, supports this explanation.

Fig. 3

Targeting Cerulean to membrane alters its fluorescence lifetime. (a) Spectral image of C-K-Ras. (d) Spectral image of EGFP-K-Ras. (c) Spectral image of Venus-K-Ras. (d) Average fluorescence lifetimes $(\mathrm{meant}\pm \mathrm{SD})$ for Cerulean $(n=11)$ , C-K-Ras $(n=12)$ , Venus $(n=18)$ , V-K-Ras $(n=20)$ , EGFP $(n=10)$ , and EGFP-K-Ras $(n=11)$ are graphed. The fluorescence lifetime of Cerulean-K-Ras is significantly different from Cerulean alone ( $p<0.05$ unpaired $t$ -test). However, the lifetimes of Venus and V-K-Ras were not different ( $p>0.05$ unpaired $t$ -test). Similar results were obtained from the EGFP constructs.

3.3.

Attenuation of the lifetime of Cerulean correlates to the amount of EM-FRET

Having demonstrated that increasing the local concentration of Cerulean can alter its lifetime, we wanted to further explore the relationship between EM-RET and lifetime attenuation observed with Cerulean. Specifically, if EM-RET could alter the fluorescence lifetime, then decreasing the separation distance between two Cerulean molecules in a protein dimer should result in a corresponding decrease in the lifetime. To test this hypothesis, we generated two additional constructs in which the two Cerulean ORFs were separated by 17 and 32 amino acids and called C17C and C32C, respectively. Photons that were emitted parallel and perpendicular to the excitation light source were simultaneously counted and correlated with the laser pulses to generate lifetime spectra. These parallel and perpendicular lifetime spectra were processed using Eqs. 2, 3 to generate florescence anisotropy and lifetime decay curves, respectively. Individual curves obtained from HEK 293 cells transfected with C $(n=7)$ , C5C $(n=6)$ , C17C $(n=6)$ , and C32C $(n=6)$ were averaged and plotted [Figs. 4a, 4b, 4c ]. All three constructs containing two Ceruleans showed EM-RET as demonstrated by the change in slope of the early part of the anisotropy decay curve when compared to C [arrow in Fig. 4a]. Note that as the separation distance between the Ceruleans increased the EM-RET decreased [Fig. 4b]. A plot of the normalized and average fluorescence lifetime decays [Fig. 4c] for C, C5C, C17C, and C32C shows that Cerulean lifetimes decreased as EM-RET increased. Therefore, increasing the separation between the two Ceruleans resulted in longer lifetime decays. These data support our conclusion that the lifetime of a Cerulean is attenuated by EM-RET.

Fig. 4

Effect of energy migration on lifetime. (a) Anisotropy of C, C5C, C17C, and C32C. Note the change in slope in the early part of the curves (arrow). (b) Anisotropy increases with increased linker size. (c) Lifetimes of C, C5C, C17C, and C32C. Note the Cerulean lifetimes decrease as a function of linker length.

3.4.

The presence of EM-FRET affects Cerulean FRET efficiencies

The decrease in Cerulean lifetime that correlates with energy migration is rather small. For example, the change in lifetime measured between C5A and C5C was approximately $100\mathrm{ps}$ , and the difference between Cerulean and Cerulean-K-Ras was approximately $300\mathrm{ps}$ . To illustrate the impact that such small changes in donor lifetime can have on a FLIM-FRET measurement, we measured the FRET efficiency of a protein domain whose crystal structure had been solved and was known to form a cytoplasmic mushroom-shaped trimeric structure as a result of subunit association through a coiled-coil motif [see Fig. 5a ]. We produced a DNA construct encoding the Traf 2 domain of the Traf protein where a Cerulean was attached to its N-terminus, and Venus was attached to its C-terminus (CTV).¹⁰ Related control constructs comprised of the Traf 2 domain attached to Cerulean and Amber (CTA) or Venus and amber (VTA) were also generated. The Traf 2 crystal structure predicts that the C- and N-termini should be separated by at least $8\mathrm{nm}$ , not including the distance added by linkers or the size of the FP $\beta$ -barrel structure ( $2.4\text{-}\mathrm{nm}$ diameter and $4\text{-}\mathrm{nm}$ high).³⁵ A conservative estimate of the total separation distance between Cerulean and Venus in CTV is $\sim 10\mathrm{nm}$ . With a Cerulean to Venus Förster distance of $5.4\mathrm{nm}$ , a Cerulean lifetime of $\sim 3\mathrm{ns}$ , and assuming a random dipole-dipole ori entation $({\kappa }^2=2∕3)$ , the predicted FRET efficiency for this fluorophore pair separated by $10\mathrm{nm}$ is 0.024 (with a FRET transfer rate of $0.008{\mathrm{ns}}^{-1}$ ). Because the Traf 2 domain forms a trimeric assembly, the three Cerulean donors are predicted to be in close proximity [see Fig. 5a]. This was verified in living cells by fluorescence anisotropy decay analysis [Fig. 5b]. A fast anisotropy decay component associated with energy migration was observed in cells transfected with CTA. A similar but more prominent drop in anisotropy was observed for VTA (data not shown). Likewise, it is expected that three acceptors should also be present at the top of the mushroom structure. Thus, for Traf 2 labeled with Cerulean and Venus, we expect that an excited Cerulean could transfer energy to not one but three potential acceptors, each approximately $10\mathrm{nm}$ away. Accounting for multiple acceptors, we revised our FRET efficiency prediction for the Traf 2 domain trimeric assembly to 0.067 based on an ensemble transfer rate of $0.024{\mathrm{ns}}^{-1}$ $(3\times 0.008{\mathrm{ns}}^{-1})$ . TCSPC was used to measure the fluorescent lifetimes of Cerulean alone, as well as Cerulean in CTV construct and in the CTA construct [Fig. 5c]. Cerulean had a lifetime of $2.94\pm 0.11\mathrm{ns}$ $(\mathrm{mean}\pm \mathrm{SD},n=10)$ , CTV had a lifetime of $2.38\pm 0.13\mathrm{ns}$ $(n=10)$ , and CTA had a lifetime of $2.59\pm 0.11\mathrm{ns}$ $(n=10)$ . In the FLIM-FRET method, the FRET efficiency is calculated by subtracting from 1 the ratio of the donor lifetime in the presence of acceptors divided by its lifetime in the absence of acceptors [see Eq. 1]. If we use the lifetime of Cerulean alone as the denominator in this equation, we calculate a FRET efficiency for CTV of $0.19\pm 0.05$ $(n=10)$ . Using the lifetime of Cerulean alone, however, fails to account for an energy migration correlated decrease in Cerulean lifetime that is expected in this construct. In contrast, if we use the lifetime of CT or CTA as the denominator, we calculate FRET efficiencies for CTV of $0.06\pm 0.02$ $(n=30)$ ¹⁰ and $0.08\pm 0.02$ $(n=10)$ , respectively. These FRET efficiency calculations do account for Cerulean energy migration. Furthermore, the fact that the FRET efficiencies of CT and CTA were not significantly different ( $p>0.05$ , Tukey’s multiple comparison test) suggests that adding amber does not alter the lifetime of CT. The estimated FLIM-FRET efficiency of CTV using the Cerulean lifetime differed from the values estimated using CTA or CT by 0.13. The FRET efficiency of CTV calculated using the Cerulean lifetime was significantly different $(p<0.001)$ from the values obtained using CT or CTA lifetimes. The FRET efficiency estimate based on using the lifetime of CTA was consistent with our predictions based on the crystal structure of Traf 2 (0.07; see discussion above). We have previously shown that a spectral method of measuring FRET efficiencies, sRET, has an accuracy of $\pm 0.05$ .²⁰ We have also previously used sRET to measure a FRET efficiency for CTV of $0.02\pm 0.07$ .¹⁰ We repeated those sRET measurements and obtained a FRET efficiency for CTV of $0.08\pm 0.06$ ( $\mathrm{mean}\pm \mathrm{SD}$ , $n=24$ ). Both of these values were consistent with the FLIM-FRET value calculated using CTA’s lifetime in the denominator.

Fig. 5

Effects of EM-RET correlated lifetime attenuation on FRET. (a) The structure of Traf 2 domain of the Traf protein, used in CTV and CTA, shows that Traf exists as a mushroom-shaped homotrimer. Alpha helices near the N-terminus (yellow arrowheads), which are attached to Cerulean in CTV and CTA, associate to form a coiled-coiled structure. The C-terminus (blue arrowheads) of the protein forms the head of the mushroom and is tagged to amber or Venus in CTA and CTV, respectively. (b) Anisotropy analysis of CTA (light blue circles) is compared to Cerulean (CER) shows that CTA exhibits EM-RET as seen from the rapid depolarization in the early part of the anisotropy decay curve. (c) Lifetime curves of cells transfected with Cerulean (C, $n=10$ ), CTA $(n=20)$ , and CTV $(n=20)$ were normalized to peak counts, averaged, and plotted. The slopes of C, CTA, and CTV are different. (d) Depicts the FRET efficiency of CTV. The FLIM-FRET efficiency of CTV calculated using the lifetime of Cerulean (dark green) is different from the value measured by sRET (blue). The use of the CTA lifetime in the FLIM-FRET calculations produced FRET efficiencies similar to those measured by sRET (light green). The ${}^{*}$ denotes FRET efficiencies for CTV calculated using the lifetime of CT as the denominator that were previously published.¹⁰

We have observed a decrease in Cerulean fluorescence lifetime in five different constructs: C5C, C17C, C32C, C-K-Ras, and CTV. The constructs C5C, C17C, C32C, and CTV have a cytoplasmic localization while C-K-Ras was membrane targeted. Thus, this change in lifetime did not correlate with a specific cellular localization. Common to cells transfected with these five constructs is the existence of a fast fluorescence anisotropy decay component. This suggests that the decrease in Cerulean lifetime is correlated with EM-RET. This phenomenon was not observed for similar constructs (V5V, V-K-Ras, and VTA) built using a yellow spectral variant of GFP, e.g. Venus. Similarly, EGFP-K-Ras also failed to express this anomalous behavior. Thus, it appears that the energy migration correlated attenuation of fluorescent lifetime is specific to the cyan chromophore found in Cerulean and formed from ${\mathrm{Thr}}_{66}\text{-}{\mathrm{Trp}}_{67}\text{-}{\mathrm{Gly}}_{68}$ but not observed in the chromophores of Venus $\left({\mathrm{Gly}}_{66}\text{-}{\mathrm{Tyr}}_{67}\text{-}{\mathrm{Gly}}_{68}\right)$ or EGFP $\left({\mathrm{Thr}}_{66}\text{-}{\mathrm{Tyr}}_{67}\text{-}{\mathrm{Gly}}_{68}\right)$ . Presumably the contribution of ${\mathrm{Trp}}_{67}$ to the chromophore is responsible for this behavior. Consistent with this hypothesis is the finding that a similar construct composed of two covalently linked CFP molecules also expressed a drop in its fluorescent lifetime. Like Cerulean, the chromophore in CFP is also formed from ${\mathrm{Thr}}_{66}\text{-}{\mathrm{Trp}}_{67}\text{-}{\mathrm{Gly}}_{68}$ . While we do not know the mechanism of this phenomenon, we speculate that this cyan chromophore might have multiple excited states, one of which has a faster decay time, and is either not populated, or poorly populated by direct excitation, but efficiently populated by energy migration.

3.5.

Conclusions

Our result differs from a previous report, which argued that the lifetime of Cerulean was not altered by the presence of EM-RET.¹⁹ One major difference between the two studies was that our experiments were performed in living cells while the other study was based primarily on spectroscopy of purified proteins. Additionally, the presence of EM-RET was not demonstrated in the other study’s Cerulean-Cerulean construct. Therefore, several plausible explanations could explain the discrepancies between the two reports, including linker proteolysis during isolation, incomplete Cerulean maturation or folding in the bacterial host, suboptimal, separation distance (27 as compared to a 5 amino-acid linker between the Ceruleans), or a dipole orientation factor with a value close to zero in the expressed protein.

While the changes in Cerulean fluorescent lifetime that were correlated with energy migration were modest, we have shown that these changes can result in significant errors when measuring FRET efficiencies using fluorescent lifetime imaging. This is particularly true for samples with low FRET efficiencies. Our findings suggest that the practice of using untagged CFP or Cerulean as a donor-alone control in FLIM-FRET analysis should be avoided if there is any possibility that multiple donors might be localized in close proximity. Under these circumstances, a conservative approach for a donor-alone sample would be to measure lifetimes from cells transfected with the same Cerulean donor construct but substituting Amber for Venus in the acceptor construct. Since Amber differs from Venus by a single point mutation, it is unlikely to alter any molecular assemblies formed between a particular Cerulean construct and a Venus construct encoding a putative interacting protein. Obviously, it is also prudent to check FLIM-FRET results with other FRET methods that are not based on lifetime measurements.