Labor intensiveness of FRET measurements in liposomes
In detergent samples, peptide and detergent molecules exchange between micelles very quickly. Therefore, to measure FRET, one can start with a sample of donor-labeled peptide in detergent and titrate a detergent solution of acceptor-labeled peptide. With highly hydrophobic peptides in liposomes, however, we cannot do titration experiments. Titrating liposomes with acceptor-labeled peptides into liposomes with donor-labeled peptides will not lead to their equilibration. Instead, each sample must be prepared separately from stocks of lipids and donor- and acceptor-labeled proteins. A new sample must be prepared for each parameter such as peptide-to-lipid ratio, donor-to-acceptor ratio, or peptide concentration. Furthermore, for each measurement, we need three different samples: (i) a sample containing both donor and acceptor, (ii) a “no FRET” control containing the donor only, and (iii) an acceptor-only control to monitor the direct excitation of the acceptor fluorophore. Therefore, FRET measurements in liposomes are much more tedious than FRET measurements in detergents.
Confirming TM orientation of the peptides in the bilayer
In detergent micelles, the detergent molecules are expected to pack around the TM domains into a spherical or ellipsoidal aggregate. Therefore, we usually do not worry about the orientation of the TM helix in the spherical micelle. In liposomes, however, one must prove that the orientation of the helix is indeed TM. Tilt of the helix with respect to the bilayer normally can be easily measured using oriented circular dichroism (OCD) in oriented multilayers prior to hydration. As shown previously, the OCD spectra for helices that are normal and parallel to the bilayer plane are dramatically different [23
]. Helices that are parallel to the membrane plane exhibit two minima at 205 and 225 nm and a maximum around 192 nm. TM helices, however, exhibit a single minimum around 230 nm and a maximum around 200 nm.
If the organic solvent used does not dissolve the two components (lipids and peptides) equally well, the helices may get “trapped” at the interface, perhaps in the form of peptide aggregates, and the OCD spectrum will reveal a mixture of TM and interfacial conformations. We have observed, however, that a mixture of HFIP/TFE/chloroform is a good solvent for both TM peptides and lipids and ensures the TM orientation of the peptides [22
When peptides and lipids are mixed in organic solvents, they can either (i) form a homogeneous mixture (i.e., a single “phase”) or (ii) segregate into two or more distinct lipid- and peptide-rich phases. The FRET method relies heavily on the assumption that the two components can be completely and thoroughly mixed to form a single phase. Therefore, homogeneity of peptide/lipid mixtures should be assessed using different methods such as X-ray diffraction, fluorescence microscopy, and FRET efficiencies as described below.
Phase separation in lipid systems is easily “diagnosed” with X-ray diffraction. A homogeneous sample gives rise to a single set of Bragg peaks. A phase-separated sample shows either (i) two sets of Bragg peaks or (ii) a single set of Bragg peaks, identical to pure lipid samples, and one or several sharp lines due to protein aggregates. Our previous work (unpublished results) has demonstrated that phase separation is particularly likely to occur in dry samples. Therefore, X-ray diffraction of dry samples provides a very stringent test for possible phase separation.
shows raw X-ray data for dry dioleoylphosphatidylcholine (DOPC) bilayers containing 5 mol% peptide. The “raw” intensity versus 2Θ plots show a single set of Bragg peaks. The relative intensities of the peaks differ substantially from intensities recorded for dry DOPC, characterized by a very large fourth peak (data not shown). Therefore, proteins and lipids appear to be thoroughly mixed and form a single phase.
Fig. 2 Raw X-ray data for dry DOPC bilayers containing 5 mol% peptide, showing a single set of Bragg peaks. No phase separation between lipids and peptides was observed using X-ray scattering. The arrows point to the positions of the first, third, and fourth (more ...)
Next we investigated whether macroscopic phase separation occurs in single bilayers containing the TM domains using fluorescence microscopy. We prepared unilamellar POPC liposomes containing Cy3-labeled TM domains and NBD–PE. Vesicles were incubated with clean microscope glass slides for 30 min, and the excess vesicles were removed via extensive rinsing with buffer, leaving a bilayer-coated glass surface. NBD–PE and TM–Cy3 were imaged using the appropriate filters [26
]. To prove that the imaged structures were single bilayers, we measured lipid diffusion coefficients using fluorescence recovery after photobleaching (FRAP). The fluorescence images were analyzed with the public domain software ImageJ, and the calculated diffusion coefficients were approximately 2 × 10−8
/s, exactly the values expected for lipid bilayers. The fluorescence images of both Cy3 and NBD appeared to be homogeneous at the micron scale [26
Aggregation of the proteins due to their dissolution from the lipid matrix can be further detected by measuring FRET as a function of acceptor concentration. It has been shown that if the helices form dimers but no higher order aggregates (such that only monomers and dimers are present), FRET depends linearly on the acceptor ratio [27
] but a larger aggregate either in or out of the bilayer will have a nonlinear dependence on acceptor concentration. In our model system, FRET depends linearly on acceptor concentration [22
] (data not shown).
After hydration, the multilamellar liposome solutions were subjected to freeze–thaw cycles, a method routinely used to achieve equilibration. We observed that after one cycle, the turbidity of the samples was obviously reduced. Such a substantial decrease in turbidity is surprising given that it does not occur for lipid MLVs that do not contain the protein. Therefore, it appears that the presence of the peptide is promoting the formation of relatively small MLVs. This is true for peptide concentrations ranging from 0.01 to 1 mol%. shows the UV absorbance (A) and the CD spectrum (B) of an MLV sample containing 0.9 mol% Cy5-labeled peptide (labeling yield ~30%) that has been cycled three times through the main phase transition. As shown in , the absorbance is relatively low, such that a CD spectrum with a high signal-to-noise ratio can be collected. Although the CD spectrum is likely affected by scattering for wavelengths less than 210 nm, the quality of the spectrum is good enough that a conclusion can be drawn with confidence that the peptides are helical. The ability to draw such a conclusion is critical because TM helices may misfold and aggregate into β-sheets. Therefore, it is recommended that the CD spectrum be recorded for each sample. As discussed above, the TM orientation in the bilayer should be confirmed using OCD.
Fig. 3 Absorbance spectrum (A, dashed line) and CD spectrum (B) of MLVs containing 0.9% peptide (optical path = 0.1 cm). The peptide was labeled with Cy5, and the labeling yield was 27%. The MLVs were freeze–thawed three times, and the two spectra were (more ...)
Having shown above that the TM peptides are homogeneously distributed when MLVs are prepared, we next assessed whether they remain homogeneous over time. We observed that the fluorescence intensity and the FRET signal of such MLVs are very stable. shows the FRET signal for rhodamine- and fluorescein-labeled TM helices. The liposome samples were freeze–thawed once, and the FRET signal was measured (solid line). After two additional freeze–thaw cycles, the fluorescence spectrum was measured again (open circles). The two spectra are identical, suggesting that the samples are fully equilibrated after only one freeze–thaw cycle. To further assess signal stability, samples were kept in the refrigerator for 1 month, after which FRET was measured and was found not to have changed.
Fig. 4 FRET spectra recorded for 0.15 mol% fluorescein-labeled peptide and 0.15 mol% rhodamine-labeled peptide in MLVs. The MLVs were freeze–thawed once, and the FRET signal was measured (solid line). After two additional freeze–thaw cycles, (more ...)
To determine whether MLVs can be used for FRET measurements, we investigated the effect of light scattering from MLVs on the fluorescence spectra such as the spectrum shown in . compares the observed intensity for two samples containing 0.3 mol% protein. One of the samples (dashed line) had unlabeled peptides and served as a “scattering control,” whereas the second sample (solid line) contained rhodamine- and fluorescein-labeled peptides (1:1 ratio). Comparison of the two spectra suggests that the measured fluorescence intensity does not have a sizable contribution due to scattering. In addition, we showed that scattering does not reduce the fluorescence intensity. shows the spectrum of an MLV sample containing fluorescein-labeled peptides (solid line). To eliminate light scattering from vesicles, Triton X-100 was added to the sample to solubilize the MLVs (decreasing the absorbance from ~0.9 to ~0.3 at 525 nm for optical path 1 cm), and the spectrum was remeasured (dashed line). The change in fluorescence intensity after the addition of Triton X-100 was insignificant. Therefore, we conclude that scattering does not affect the fluorescence intensity in our experimental system, so that MLVs can be used to measure FRET in vesicles.
Fig. 5 Contribution of scattering to the measured FRET spectra. Solid line: MLVs containing 0.1 mol% fluorescein-labeled peptide and 0.1 mol% rhodamine-labeled peptide. Dashed line: MLVs containing 0.2 mol% unlabeled peptide. The latter sample serves as a control (more ...)
Fig. 6 Fluorescence intensity of fluorescein-labeled peptides in MLVs before (solid line) and after (dashed line) the addition of Triton X-100. Lipid concentration was 1 mg/ml, and the sample contained 0.04 mol% fluorescein-labeled peptide. Triton X-100 was (more ...)
Finally, we demonstrated that the FRET efficiency in MLVs is determined only by the protein-to-lipid ratio, not by the total peptide and lipid concentrations (see ). Results are discussed in detail below.
Fig. 8 (A) FRET signal for two MLV samples with the same peptide-to-lipid ratio but different buffer volumes. A common lipid-to-protein stock was prepared to ensure an identical peptide-to-lipid ratio. The stock was divided into two samples of volumes 50 and (more ...)
Large unilamellar vesicles
The LUV is a system that is widely used as a model for measurements of protein binding to membranes and insertion into membranes [29
]. Therefore, we sought to assess the relevance of this system for FRET measurements of TM helix dimerization. To prepare LUVs, we extruded the MLV samples through a 100-nm pore membrane. To calculate FRET in liposomes, both donor-only and donor–acceptor samples were extruded and FRET was calculated according to Eq. (1)
. As shown in , we observed no statistically significant decrease in FRET in LUV compared with MLV. Therefore, FRET efficiencies and helix–helix interactions measured in MLVs and LUVs are comparable.
Fig. 7 Measured FRET efficiencies after a single freeze–thaw cycle (black), after 1 month equilibration (gray), and subsequent extrusion to produce LUVs (white) for three samples. Sample A: 0.1 mol% fluorescein-labeled peptide and 0.1 mol% rhodamine-labeled (more ...)
A potential problem with extrusion is the loss of proteins and lipids in the extrusion process. This is why protein and lipid concentrations are generally different from the intended ones and must be measured after extrusion. We determined protein concentration in the LUVs by measuring dye absorbance or fluorescence and the lipid concentration using a standard phosphate assay (). We found that typical losses of proteins and lipids were identical; that is, between 13 and 18% of both lipids and proteins were lost during the extrusion process. Because the percentage losses were similar, the protein-to-lipid ratio remained the same (). Despite material loss, one can still do quantitatively meaningful experiments if only the protein-to-lipid ratio, but not the water content, is the determinant of the FRET efficiency. Therefore, we expanded the MLV study in to extruded vesicles. We started with a common lipid/protein solution in organic solvent (Cy3-to-Cy5 ratio of 1). Then we added different amounts of buffer to identical dried aliquots of the lipid/peptide stock, and we collected fluorescence spectra for various buffer dilutions. In , we plot the ratio of the acceptor-to-donor peaks (Cy5 to Cy3) as a measure of FRET for two different samples (different protein-to-lipid ratios). The Cy5/Cy3 amplitude does not change for dilutions up to 32 times. Therefore, FRET does not depend on the buffer volume; rather, it depends only on the protein-to-lipid ratio.
Loss of peptides and lipids during extrusion
The observation that the FRET efficiencies depend solely on the protein-to-lipid ratios has an important implication. It suggests that we can directly probe the monomer/dimer protein equilibrium in the bilayer. In liposomes, the protein acts as a solute and the lipid matrix acts as a solvent, whereas water is a separate phase that has no effect on the effective concentration of peptide in the membrane. Therefore, FRET measurements in liposomes allow us to extract “absolute” thermodynamic parameters pertaining to dimerization of TM helices in lipid membranes.
Small unilamellar vesicles
SUVs were produced via sonication of MLVs. When we attempted to measure FRET in SUVs, we noticed a decrease in the amplitude of the fluorescence spectra. This result is shown in , where two of the spectra, shown with the dashed and dotted lines, were acquired in 5 and 10 min, respectively, after the first spectrum (solid line). In this experiment, the cuvette was not moved and the sample was not manipulated in any way between spectra acquisitions. We note that such a decrease in signal amplitude, characteristic of SUVs, is not observed for MLVs or LUVs. Judging by the shape of the spectra (i.e., by the ratio of Cy3-to-Cy5 fluorescence), the FRET efficiency does not change as the amplitude decreases. SUVs are known to be far from equilibrium, and we believe that the SUVs are aggregating and coming out of solution. Although the exact mechanism is unknown, our results suggest that SUVs are not an appropriate system for FRET measurements, particularly if Eq. (1)
is used to calculate FRET efficiencies.
Fig. 9 Fluorescence spectra of Cy3- and Cy5-labeled peptide mixtures in SUVs. Two of the spectra (dashed line and dotted line) were acquired 5 and 10 min, respectively, after the first spectrum (solid line). The amplitude of the signal decreased steadily over (more ...)