5. RESULTS
5.1 MAIN PHASE TRANSITION OF BIOMEMBRANES
5.1.3 TIME-RESOLVED FLUORESCENCE
Time-resolved fluorescence spectroscopy measurements were performed to obtain a better understanding of the molecular level processes underlying the steady state fluorescence data. Pyrene monomer lifetimes (τM) and excimer formation (τR) and decay times (τD) were measured for PyrPC in DPPC and DNPC in the vicinity of Tem. As expected, the integrated excimer intensity IntIe of PyrPC time-resolved fluorescence emission at 480 nm displayed similar behavior as the corresponding steady state Ie. For both DPPC and DNPC, PyrPC excimer fluorescence was best described by two risetimes (7-72 ns). For PyrPC residing in DPPC, the fractional intensity of the shorter risetime (~10 ns) was maximally approximately one percent so that only the longer risetime was significant. Similarly, the fractional intensity of the shorter risetime of PyrPC in DNPC declined steeply in the first temperature region, where after it was a minor component.
Excimer emission of PyrPC exhibited a single decay time in DPPC (57-95 ns) and two decay times in DNPC (10-85 ns), whereas the monomer fluorescence emission at 398 nm was characterized by two decay times for both matrixes, expressed as the weighted average lifetime (τ M). The components of τ M varied between 7-16 and 73-119 ns for DPPC and 10-57 and 64-105 ns for DNPC LUVs. The fractional intensity of the shorter monomer decay time for PyrPC in DPPC was maximally 7%, while for PyrPC in DNPC, the amplitudes of the monomer decay times were approximately equal.
Figure 10. Weighted average decay time τ Mversus (T-Tem) for monomer emission obtained from time-resolved data for PyrPC in DPPC LUVs with XPyrPC = 0.02.
Figure 11. Excimer emission risetime τR using Savitzky-Golay filtering with a window of two data points.
For PyrPC in DPPC, τ M displayed no significant anomalies in the vicinity of Tem, and an almost steady overall decrement by 37 ns in the pyrene monomer lifetime was observed, when (T-Tem) was increased from -10° to 5° (Fig. 10). In contrast, the data for PyrPC in DNPC revealed notable fluctuation in the monomer lifetime and an overall decrease in τ M within the temperature range studied (Study II, Fig. 6).
In the first temperature region, excimer risetime τR for PyrPC in DPPC decreased with increasing temperature from ~49 ns to ~45 ns (Fig. 11 A), in keeping with enhanced
45
collisional rate due to augmented lateral diffusion of PyrPC in the PyrPC clusters (Somerharju et al., 1985; Chong et al., 1994). Similar behavior was observed for both risetimes for PyrPC in DNPC (Fig. 12). The shorter risetime of ~25 ns for PyrPC in gel phase DNPC indicates that the excimer formation is not limited by diffusion only, but also by additional rearrangements in the packing of the clustered pyrenyl moieties. It is feasible that some population of static dimers is involved. The softening of the gel phase bilayer with increasing temperature occurred simultaneously with a decrease in the fractional intensity of the shorter risetime component IR1. Accordingly, a decrease from the maximum ~0.43 at (T-Tem) ~ -20° to ~0.02 at (T-Tem) ~ -9° was observed. This suggests that the pyrene probes transfer from the gel phase clusters into the growing line defects. The above changes are in keeping with pretransition of DNPC starting at (T-Tem) ~ -10°, possibly involving pseudo one-dimensional corrugations of fluid lipids (Heimburg, 1998; 2000). Excimer fluorescence decay times for PyrPC in DNPC converged within the pretransition region (Fig. 12), whereas for PyrPC in DPPC excimer decay decreased steadily in the first temperature region from ~95 to ~89 ns.
For PyrPC in DPPC, the values for excimer risetime τR increased pronouncedly from ~46 to ~60 ns in the second temperature region, and a minor decrement in the integrated intensity of excimer emission occurred (Fig. 11). This complies with PyrPC being distributed into the interfacial boundary separating the coexisting gel and fluid phases. The mode of lateral diffusion determining the rate of excimer formation should thus change from 2-dimensional to more 1-dimensional. Concomitantly, the value for τD
continued to decrease progressively without notable changes in monomer decay. This is in keeping with a temperature-induced acceleration in the excimer decay and an increase of fluid-like domains at the expense of gel-like domains in region II. Interestingly, IntIe for PyrPC in DNPCdecreased steeply already in the second temperature range, whereas the two excimer decays continued to converge, and no significant changes in the excimer risetimes were observed (Fig. 12).
The disparity in the fluorescence properties of PyrPC between the matrixes is explained by the difference in the effective length and saturation of the acyl chains of DPPC and DNPC. Accordingly, for DNPC, trans → gauche isomerization of the terminal ends of the acyl chains easily allows this lipid to fill the voids underneath the shorter PyrPC,
while this is not possible for DPPC. As a result, the fluorescence quenching by water and the perturbation of the neighboring acyl chains by the bulky PyrPC are dimished, allowing a more random distribution of the probes in the gel-fluid interface of DNPC.
Temperature region III coincides with the pronounced reduction in the acyl chain order signaled by DPHPC anisotropy and a significant fraction of the transition enthalpy for both DPPC and DNPC LUVs. The kinetics of excimer formation and decay were markedly changed, while no significant variation in τ M for PyrPC in DPPC was observed.
The values for τ M for PyrPC in DNPC continued to fluctuate, and a local maximum of ~84 ns at (T-Tem) ~2° was reached. The decreasing values for IntIe and the quantum yield of fluorescence together with an almost steady τR and τD –levels for PyrPC in DPPC, suggest that the probes become maximally separated, after being released from the disappearing domain boundaries. These changes are in keeping with the formation of a time-averaged regular distribution of the probe molecules (Chong and Sugár, 2002) in DPPC in the temperature region III (Fig. 8 and 11).
The decrease in IntIe versus (T-Tem) indicates that the redistribution of PyrPC starts at lower temperatures in DNPC LUVs compared to DPPC. The pronounced increase and the local maximum of ~72 ns at Tem for τR2 together with the nearly vanishing difference between τD1 and τD2 in the temperature region III are consistent with the intermediate phase occurring also in the course of DNPC main transition. For DNPC, the formation of the intermediate phase appears to start at (T-Tem) → -1°, this phase prevailing up to (T-Tem) ~5°. Importantly, for both matrixes, the melting of the acyl chains, detected by DPHPC anisotropy and enthalpy change, was completed in region III.
It thus seems that the transition for PCs starts as a first-order process with gel and fluid phase coexistence and develops into a second-order transition with further increase in temperature, due to increasing heterophase fluctuations.
Figure 12. Excimer fluorescence emission fractional intensities IR1 (○) and IR2 (■) of the risetimes (panel A), and decay times ID1 (○) and ID2 (■) (panel C) as well as variation of the excimer risetimes (panel B), and decay times (panel D) as a function of temperature.
In the temperature region IV, the time-resolved fluorescence of PyrPC signals the bilayer to be homogenous for both DPPC and DNPC. For PyrPC in DPPC, a steady decrease in τR, τD, and τ M was observed together with a modest increase in IntIe, in keeping with thermally induced increase in the lateral mobility.
For PyrPC in DNPC and at (T-Tem) > 5o, the fractional intensity of the excimer decay component ID1 increased to unity, and the decay was described by a single process
-25 -20 -15 -10 -5 0 5 10 15 20 25
(Fig. 12). Furthermore, the weighted monomer decay time τ M for PyrPC in DNPC started to decrease, and the fractional intensities of IR1 and ID1 remained nearly constant.
There are no anomalies in the time-resolved data or enthalpy that would require distinction of the temperature region V for DNPC. Yet, Im of the steady-state data (Fig. 9) was anomalously reduced starting at (T-Tem) ~10°. This necessitates region V and indicates that the local environment and the lateral mobility of the probe are slightly modified also in the fluid phase with increasing temperature.
5.1.4 CONFOCAL FLUORESCENCE MICROSCOPY RESULTS
Confocal fluorescence microscopy was undertaken in order to investigate the lack of two-phase region in the course of the main transition, indicated by the fluorescence spectroscopy measurements. Giant DNPC unilamellar vesicles containing the fluorescent phospholipid analog NBDPC (XNBDPC = 0.02) were studied by cooling scans between -3°
< (T-Tem) < 6°, with Tem derived from the cooling scans for DNPC LUVs (Ø ~ 0.2 µm) (Fig. 13).
In keeping with the fluorescence spectroscopy measurements, the microscopy data revealed that the lateral distribution of the NBDPC probe in DNPC GUVs remained homogenous down to (T-Tem) ~ -0.5° (i.e. 20.5°C). When (T-Tem) ~ -0.5° was reached, black gel phase domains appeared, and the fluorescent probes tended to cluster along the edges of the emerging domains (Study IV, Fig. 3). At this temperature, GUVs also began to detach from the Pt wire.
The relative size of the dark domains increased with further decrease in temperature to (T-Tem) ~ -1°. The appearance of the two-phase region was preceded by a modest shrinking of the vesicles starting at (T-Tem) ~ 1° (Fig. 14). The gradual decrease in the vesicle diameter depended on the initial size of the GUV, being ~25% for the middle sized GUVs, diameters in the range from 15 to 25 µm, and ~15% for the smallest GUVs, diameters ranging from 5 to 15 µm (Fig. 14).
Interestingly, most of the vesicles eventually collapsed in the gel-fluid coexistence region before reaching homogeneous gel phase (Fig. 13, panel C). The
rupture of the DNPC GUVs in the course of the cooling scan is consistent with the gel phase domains starting to grow with a concave curvature. This involves unstabilizing changes in the relative magnitudes of the elastic deformation variables i.e. line tension, bending moduli, and Gauss moduli (Baumgart et al., 2005). A decrease in the vesicle diameters by ~7-8% were reported for the main transition of DMPC and DPPC GUVs (Needham and Evans, 1988; Bagatolli and Gratton, 1999). In these experiments, however, no concomitant rupture of the vesicles was observed.
Figure 13. Confocal microscopy images for DNPC/NBDPC (0.98/0.02) GUV observed upon cooling, with Tem = 21°C defined as the heat capacity maximum measured from the exotherm of the corresponding LUVs (extruded through 0.2 µm filters).
Panel A: (T-Tem) = 4°, panel B: (T-Tem) = -0.5°, panel C: (T-Tem) = -1.5°.
Upon heating of the intact vesicles, the GUVs became homogenous at T ~26°C, this temperature coinciding with the Tem measured for the heating scans of the corresponding LUVs by DSC. Yet, only modest recovery of the GUV diameter was observed (~73%), indicating loss of lipid during the cooling scan.
Figure 14. Change of diameter (µm) of five DNPC/NBDPC (0.98/0.02) GUVs as a function of temperature upon cooling.
18 20 22 24 26 28 30
12 15 18 21 24 27 30 33 36
Diameter,µm
T, oC
5.2
CHOLESTEROL INTERACTION OF BIOMEMBRANES
5.2.1 OVERVIEW OF THE EXPERIMENTS
The hypothesis was that the colocalization of Chol with PC and SM depends primarily on the effective length of the phospholipid in question and the hydrophobic thickness of the surrounding lipid matrix. Accordingly, Ie/Im –ratios for the fluorescent lipid derivatives PyrSM and PyrPC (X = 0.01) in DMPC and DNPC were measured as a function of increasing temperature and Chol content (XChol = 0.01-0.05). Subsequently, RET from PyrPC and PyrSM to NBDchol (XNBDchol = 0.01) in DMPC and DNPC matrixes were compared. The results are expressed as the colocalization parameter C. Colocalization data for NBDchol were verified by using a less perturbing Chol analog, diBrChol (XdiBrChol = 0.01). This brominated, collisional quencher of fluorescence lacks the fluorophoremoiety. Unlike RET that may occur over considerably long distances (~10-100 Å) and includes quenchingof the donor also in the adjacent leaflet of the bilayer, collisional quenching requires actual molecular encounters. Theimpact of the different lipid compositions on the thermotropic phase behavior of the studied membranes was assessed by using DSC.
5.2.2 DIFFERENTIAL SCANNING CALORIMETRY
Heating scan measurements were performed for pure DMPC and DNPC MLVs and LUVs as well as for liposomes containing lipids used in the fluorescence spectroscopy experiments (Study III, Table 1). Pure DMPC MLVs displayed a pretransition at ~14°C and a main transition at ~23.7°C. For pure DNPC MLVs and LUVs, only the main phase transition endotherms were observed at ~26.9°C and ~26.7°C, respectively. This is in keeping with previous data showing that the pretransition enthalpy peak for the longer acyl chain PCs merges with the main transition endotherm (Jørgensen, 1995).
A single endotherm at ~24.1°C was detected also for DMPC LUVs, since extrusion abolishes the pretransition peak from the DSC trace. The impact of the probes on DMPC endotherms was similar to that reported for DPPC and DNPC liposomes containing the probes. When the MLVs contained one or more of the lipid probes (X = 0.01-0.02), pretransition was absent, and thetotal enthalpy decreased suggesting reduced trans gauche isomerization in the course of the transition.This is likely to reflect the chain disordering effect of theprobes below Tem.
5.2.3 EFFECT OF CHOLESTEROL
First, Ie/Im –values for PyrPC and PyrSM were measured for both matrixes without Chol being present. For PyrPC in DMPC, Ie/Im increased steadily upon heating, consistent with increased lateral diffusion (Hresko et al., 1986; Lehtonen and Kinnunen, 1995). As reported, the main transition of DMPC was preceded by a local maximum (denoted by T*
in Fig. 15 A) in Ie/Im for PyrPC, startingat (T-Tem) ~ 4.5° and peaking at ~1.5° below Tem. This peak was suggested to represent lateral enrichment of the probeinto the boundary between the gel- and fluid-like regions (Jutila and Kinnunen, 1997). The present time-resolved data, measured for PyrPC in DPPC and DNPC, support this view. Just below Tem, a decline in Ie/Im was observed, and the values for Ie/Im returned close to the ascending baseline. The slope of Ie/Im versus (T-Tem) for PyrPC in DMPC was, however, steeper above Tem compared to the slope below Tem, indicating that the thermally activated increase in therate of lateral diffusion in the gel and fluid phases are not equal.
In the studied temperature range, the Ie/Im –ratio for PyrSM residing in DMPC liposomes was significantly lower than that measured for PyrPC, and the transient peak in Ie/Im was absent (Fig. 15 A). This could reflect slower lateral diffusion of the former probe as well as interdigitation of the pyrene moiety of PyrSM into the adjacent bilayer leaflet (Somerharju et al., 1985;Hresko et al., 1987).
Because of the significantly higher Tem of PyrSM compared to PyrPC (~43 and
~15°C, respectively), the effective length of PyrSM should exceed that of PyrPC in this temperature range, resulting in interdigitation and reduced Ie/Im –ratio (Somerharjuet al.,
Figure 15. (A) Ie/Im versus (T-Tem) for PyrPC ( ) DMPC acyl chain disordering by PyrPC is more pronounced than that of PyrSM (Study III, Table 1).
The main transition of DMPC LUVs detected by PyrSM fluorescence was manifested by a decrease and a local minimum in Ie/Im (Fig 15). Unlike for PyrPC in DMPC, a steady increase in Ie/Im was observed both below and above the slope, and the dip with the minimum at ~Tem occurred on a slightly wider temperature range.
Interestingly, the Chol content (XChol = 0.01-0.05) had a minor effect on the Ie/Im–values both below and above Tem (Study III, Fig. 2 A). This suggests that at XChol ≤ 0.05, Chol has an insignificant impact on the lateral diffusion of the pyrene-labeled probes in DMPC.
Apart from T > Tem when the Ie/Im -ratios were approximately equal for both probes, the values for Ie/Im for PyrSM in DNPC were slightly lower than those observed for PyrPC (Fig. 15 B). Compared to DMPC, in DNPC the starting levels of the Ie/Im
ratios were approximately one order of magnitude greater. Although lateraldiffusion of the probes could be higher in DNPC, it is possible that interdigitation and tilting of pyrene chains reduce the Ie/Im –values in DMPC. For the thicker DNPC bilayer, inclusion of Chol causeda pronounced and progressive decrease in Ie/Im for both probes below Tem
(Study III, Fig. 2 B). Above Tem, no changesin Ie/Im for either probe were observed by increasing the Chol content. This is consistent with colocalization of Chol and the pyrene derivatives in gel phase clusters.
5.2.5 FLUORESCENCE RESONANCE ENERGY TRANSFER
For PyrPC in DMPC, the values for Ie/Im increased, and the transient peak at T* vanished when NBDchol (XNBDchol = 0.01) was included into the LUVs (Fig. 16 A). This aligns with monolayer and fluorescence studies suggesting that Chol reduces line tension in the interface (Weis and McConnell, 1985; McConnell, 1991;Hwang et al., 1995; Mouritsen et al., 1995) by localizing between the coexisting solid and fluid domains (Jutila and Kinnunen, 1997). It is likely that for PyrPC in DMPC LUVs, the increment in Ie/Im
induced by NBDchol reflects a more efficient quenching of PyrPC monomer by this acceptor compared to PyrPC excimer. In contrast, for PyrSM in DMPC, NBDchol lowered the values for Ie/Im significantly throughout the temperature range. No increase in Ie/Im up to (T- Tem)~1.5° was observed, where after the ratio increased almost linearly (Fig. 16 A).
For PyrPC in DNPC matrix, the impact of NBDchol on the temperature dependency of Ie/Imwas less prominent than that observed for PyrPC in DMPC. When T
< Tem, the starting level of the Ie/Im -ratio was lower, and the values for Ie/Im declined, reflecting the gel-to-fluidmelting processes (Fig. 16 C). The RET acceptor NBDchol had a minor effecton the Ie/Im -ratio measured for PyrSM in DNPC (Fig. 16 C).
Colocalization parameter C for PyrPC and NBDchol in DMPC, revealed first dimished colocalization in the gel phase with increasing temperature up to a local minimum at (T-Tem) ~ -5°, followed by an increase and a local maximum at ~T* (Fig. 16 B). Another local minimum occurred at ~Tem with a subsequent modest increase in C.
Figure 16. Values for Ie/Im versus (T-Tem) for DMPC LUVs incorporating PyrPC ( ) or PyrSM ( ) (X = 0.01) together with NBDchol (X = 0.01) (A). Colocalization parameter C versus (T-Tem) defined for the PyrPC- ( ) or PyrSM- ( ) NBDchol RET-pair (B). Values for Ie/Im versus (T-Tem) for DNPC LUVs incorporating PyrPC ( ) or PyrSM ( ) (X = 0.01) together with NBDchol (X = 0.01) (C). Colocalization parameter C versus (T-Tem) defined for the PyrPC- ( ) or PyrSM- ( ) NBDchol RET-pair residing in DNPC (D).
In contrast, C versus (T-Tem) for PyrSM and NBDchol in DMPC increased steeply up to (T-Tem) ~ -2.5°. This temperature closely corresponds to the point where the Ie/Im – ratio for PyrSM starts to decrease. This data suggests that similarly to PyrPC, also PyrSM and NBDchol colocalize into the domain boundary in the two-phase region. A local
minimum in C occurred at ~Tem, where after the values for C increased. This increment in C is in keeping with thermally induced increase in the rate of lateral diffusion.
Interestingly, C for PyrPC and NBDchol in DNPC was higher than C for PyrSM and NBDchol, indicating a more effective colocalization of the former lipids within the studied temperature range (Fig. 16 D). For PyrPC and NBDchol RET-pair and upon approaching Tem, C increased modestly until a local maximum at (T–Tem) ~ -2° was reached. Then, a local mimimum occurred at ~Tem, and further increase in T had a minor effect on C (Fig. 16 D).
For PyrSM and NBDchol in DNPC and at (T-Tem) < -5°, C remained nearly unchanged. Starting at (T-Tem) ~ -5°, a decrease in C was observed without a preceding transient peak. When T > Tem, the values for colocalization parameter fluctuated around a relatively steady horizontal baseline.
5.2.6 COLLISIONAL QUENCHING OF PYRENE EXCIMER EMISSION
Colocalization parameter C defined for PyrPC and diBrChol in DMPC, revealed progressively diminished close range colocalization with increasing temperature (Fig. 17 A). At temperatures above the local minimum at Tem, C first increased steeply, and then at (T-Tem) ~ 1° a relatively steady level was reached.
For PyrSM and diBrChol, C versus (T-Tem) displayed efficient close range colocalization throughout the temperature range with a slight increase in C below Tem (Fig. 17 A). At approximately Tem, a minor local minimum occurred, followed by modest increment in C with further increase in T.
For PyrPC and diBrChol in DNPC, the trend for the colocalization parameter was very similar to that defined for PyrPC and NBDchol in DNPC. Accordingly, PyrPC and diBrChol colocalized efficiently, especially at T < Tem (Fig. 17 B). At (T-Tem) ~ -2°, a steep decrease in C occurred with a minimum at ~Tem. Subsequent increase in temperature resulted in the fluctuation of the values for C with insignificant change in the overall colocalization.
For PyrSM and diBrChol in DNPC, the C versus (T-Tem) -trace displayed lower C –values compared to PyrPC and diBrChol(Fig. 17 B). The close range colocalization for the former composition increased modestly when (T-Tem) < -7°, where after a gradual decline up to Tem was observed.
Figure 17. (A) Colocalization parameter C versus (T-Tem) defined for PyrPC- ( ) or PyrSM- ( ) 5,6-dibromo-cholestan-3ß-ol collisional quencher pair in DMPC matrix. (B) Colocalization parameter C versus (T-Tem) defined for the PyrPC ( ) or PyrSM ( ) with 5,6-dibromo-cholestan-3ß-ol collisional quencher pair residing in DNPC matrix.
-15 -10 -5 0 5
0.3 0.4 0.5 0.6 0.7 0.8 0.9
A
C
T-Tem
-15 -10 -5 0 5 10
0.2 0.3 0.4 0.5 0.6 0.7 0.8
B
C
T-Tem
6 . D I S C U S S I O N
6.1
EFFECTS OF HYDROPHOBIC MISMATCH
6.1.1 MODEL OF THE MAIN TRANSITION
The present, quasistatic transition data indicate that the main transition of PC liposomes is a sequence of transitions rather than a two-state process. Accordingly, the fluorescence spectroscopy data reveals a lack of “large-scale” coexistence of gel and fluid phases at temperatures above Tem of DPPC and DNPC LUVs. This finding is supported by the
The present, quasistatic transition data indicate that the main transition of PC liposomes is a sequence of transitions rather than a two-state process. Accordingly, the fluorescence spectroscopy data reveals a lack of “large-scale” coexistence of gel and fluid phases at temperatures above Tem of DPPC and DNPC LUVs. This finding is supported by the