- Email: [email protected]
Kinetics of lipid phase changes
Kinetics of lipid phase changes Martin Caffrey and Anchi Cheng The Ohio
State University,
Columbus,
In the past two years, the kinetics of transitions
involving
USA
the assorted lamellar
and inverted hexagonal and cubic phases in bulk hydrated lipid systems have been established
using a variety of physical techniques.
kinetic data have lead to a transition
Current
Opinion
mechanism
in Structural
Biology
Introduction
In several cases, the
being deciphered.
1995,
5:548-555
learn why nature endows biomembranes enormous lipid-type diversity.
In the context of the living cell, the lipid bilayer can be viewed as recruiting proteins and other molecules for the purpose of generating a selectively permeable, signal transducing supramolecular aggregate known as the biological membrane. The lipid component of membranes, and indeed of other lipid-rich bioaggregates, has the inherent capacity to adopt a variety of so-called liquid crystalline states, or mesophases, more ordered than the liquid but less so than the crystalline solid (Fig. 1). The lipid bilayer represents one such state, referred to as the lamellar or smectic phase. The remainder are grouped together in the so-called non-bilayer phase category. The particular liquid crystal phase accessed by manipulating the conditions depends on temperature, pressure, lipid concentration and composition of the aqueous dispersing medium. Interestingly, the majority of isolated membrane lipids are content to form one or other of the non-bilayer phases at or close to physiological conditions. In the course of undergoing such bilayer-+non-bilayer phase changes, intermediates, reminiscent of structures proposed to appear transiently during the ubiquitous process of membrane fusion, are likely to develop. This accounts, in part, for the interest in lipid-based phase-transition kinetics and mechanism studies. One way of deciphering mechanism is to establish the kinetic parameters of the transition. It is such on-going endeavors that will be covered in this review. A long-term objective in this area is to establish how lipid molecular structure and composition in mixed lipid systems affect phase-transition kinetics and mechanism. Such insights will provide for rational design strategies aimed at effecting control over not only membrane structure and function in intact organisms, but also over performance characteristics in reconstituted and formulated systems. Along the way, we hope to
with such an
To consider the area of lipid phase transition kinetics a burgeoning field is likely to be an overstatement of the case: probably 10 to 20 groups worldwide are active in the area. This pales in comparison with other areas within the realm of structural biology, although the numbers make a little more sense when viewed in the context of the complexity of the systems being studied, the sophisticated measurements needed to extract useful information and the techniques currently available for their investigation. This review, which amounts to a snapshot in time of what is happening in the field, gives a flavor of the enormous variety that exists in the systems actively under investigation and the techniques being used for the purpose. The review is of limited scope in that it covers only the period from January 1993 onwards and the literature dealing with experimental aspects of relatively fast phase transitions in bulk hydrated lipid systems. Because of the narrow focus, it has been possible to include in the review all relevant published work that has come to our attention.
Pressure-induced
transitions
Transitions
the lamellar
involving
phase using
pressure jump
The kinetics and mechanism of the barotropic (pressuresensitive) lamellar gel (Lbr)+lamellar liquid crystal (L,) phase transition in fully hydrated 1,2-dihexadecylsn-glycero-3-phosphoethanolamine (DHPE) has been studied using time-resolved X-ray diffraction (TRXRD) [l”]. The phase transition was induced by pressure jumps of varying amplitudes in the pressurization and
Abbreviations DHPE-l,2-dihexadecyl-sn-glycero-3-phosphoethanolamine; tit,-inverted
hexagonal
L,-subgel
L,-lamellar
phase; MLV-multilamellar Pn3m
548
phase;
and la3drubic
liquid
DPPC-l,2-dipalmitoyl-sn-glycero-3-phosphocholine;
crystalline
vesicle;
P-pressure;
space groups;
0 Current
phase;
Lg-lamellar
l-temperature;
Biology
gel phase;
PC-phosphatidylcholine; TRXRD-time-resolved
Ltd ISSN 0959-440X
L~S-lamellar
gel phase with
PE-phosphatidylethanolamine; X-ray diffraction.
tilted
chains:
Kinetics of lipid phase changes Caffrey and Cheng
depressurization directions at controlled temperature dieacted X-rays (78°C). Both 1ow- and wide-angle were recorded simultaneously in real-time which allowed for the direct and quantitative characterization of the long-range (lamellar repeat spacing) and short-range (chain packing) structural order of DHPE during a kinetic experiment. The image-processed live-time X-ray diffraction data were fitted using a non-linear least squares model and the parameters of the fits were monitored continuously throughout the transition. The barotropic transitions from the L, to the LB’ phase phase were two-state and from the LB’ to the L, (no formation of intermediates apparent during the transition) to within the sensitivity limits of the method. The corresponding transit time (the time during which both phases coexist) associated with the long- and shortrange order of the pressurization-induced L,+Lp phase transition decreased to a limiting value of approximately 50 ms with increasing pressure-jump amplitude. This limiting value was close to the response time of the detector/recording system. Thus, the intrinsic transit time of this transition in fully hydrated DHPE at 78°C was less than or equal to 50 ms. In contrast, the depressurizationinduced Lbt+L, phase transition was slower, taking approximately 1 s to complete, and occurred with no obvious dependence of the transit time on pressure-jump amplitude. In the depressurization-jump experiment, the lipid responded rapidly to the pressure jump in the LB phase up to the rate-determining Lp+L, transition, by converting to a low-pressure form of the LB’ phase. The latter, enigmatic behavior, suggestive of a transition intermediate, is wont to complicate the interpretation of phase transition kinetic measurements and is worthy of note by researchers in the area. Pressure jump has also been used to study the kinetics of the lamellar/inverted hexagonal (HI,; Fig. li) phase in lipids using TRXRD with a resolution of 9 ms (M Kreichbaum et al., Biopllys J 1993, 64:A296). With pressure-jump amplitudes in excess of 1 kbar the transition was completed within the time resolution of the measurement. A slower transition was observed with smaller amplitude jumps.
Lamellar
gel/liquid
crystal phase transition
in binary
lipid
mixtures
A powerful stationary relaxation technique employing volume perturbation calorimetry has been used by Biltonen and Ye ([2]; see also references therein for seminal work in this area) to study the kinetics and mechanism of the chain order/disorder transition in binary (two component) lipid membranes. In this technique, the sample housed under essentially adiabatic conditions is subjected to a small periodic change in volume by means of a piezoelectric crystal stack. The pressure and temperature response of the sample is monitored continuously during the course of the measurement performed over several decades of perturbation frequencies. The impulse-response data are analyzed in the frequency
domain, from which can be obtained the relaxation rate and insights into transition mechanism. When applied to relatively simple binary phosphatidylcholine (PC) bilayer mixtures undergoing the chain order/disorder transition, four results were found. First, the relaxation process is considerably more complex than that observed with single component systems and consists of more than a single exponential decay. Second, the relaxation rate for the mixture is at least an order of magnitude higher than that for single component membranes. Third, the relaxation rate enhancement was pronounced when the minor component of the lipid mixture had the higher temperature for the order/disorder transition. Fourth, the relaxation time versus temperature profile had two maxima regardless of whether the corresponding equilibrium heat capacity function had one or two peaks. These results suggest either that such mixed-lipid membranes exhibit a form of dynamic phase separation in the bilayer plane and/or that molecular diffusion plays an important function in defining the kinetics of in-plane phase separation.
Lamellar
gel/liquid
single component
crystal partial
phase transition
in a
system
In order to extend the volume perturbation calorimetry approach described above, an instrument has been built that allows for an analogous perturbation to be imposed on the sample with the added feature of being able to monitor structure changes directly by means of simultaneously performed TRXRD. Accordingly, we have used synchrotron-based TRXRD to monitor both low- and wide-angle difhaction changes during a partial L,/lamellar gel (LB) mesophase transition in fully hydrated monoelaidin (a monoacylglycerol with an 18-carbon nuns-unsaturated acyl chain) multilamellar vesicles in response to small pressure oscillations applied over four decades in frequency (A Cheng, M Caffrey, abstract in Biophys J 1995, 68A96). The sinusoidal pressure oscillation amplitude was small enough to elicit a linear response. The response amplitude and phase shift were both determined as a function of perturbation frequency. The fractional phase conversion response spectra for the two structure changes monitored (mesophase lamellarity and chain conformation) were very similar, indicating that the chain order/disorder transition correlates well with the lamellar repeat spacing change. The data are consistent with a transition best described by the Kolmogorov-Avrami kinetic model [3], having an effective dimensionality close to unity (0.78 f 0.05 and 0.9 kO.1 for the low- and wide-angle data, respectively) and a structural relaxation time of 13 f 2 and 10 f 3 s for the low- and wide-angle data, respectively. We have interpreted the results in the context of a bidirectional layer-by-layer transition mechanism for the La/L8 phase change. Although the transition is predominantly interface controlled, material diffusion in the form of water flux is likely to play a modest role in the phase interconversion.
549
550
lipids
(B) lamellar gel (LB)
(A)sub-gel0-d
, ripple wavelength
(E) chain packing
(C) lamellar gel-tilted (Lv)
(F) lamellar liquid crystal (L,)
d,=d,,=a.cos
(G) normal hexagonal
d hex
(HI)
(H) lamellar liquid crystal (L,)
(1)inverted hexagonal (HII)
T
T
dhex=do,=
d her
3, 2
w
(J) Cubic Pn3m (Q224)
(M) tubule
(K) Cubic la3d (Q230)
(N) unilamellar vesicle
(L) Cubic lm3m (Q22g)
(0) multi-lamellar
vesicle
Kinetics of lipid phase changes Caffrey
An X-ray-induced
transition:
lamellar/hexagonal
phase transition The X-radiation induced partial L, +HII phase transformation was observed in a fully hydrated DHPE sample at 79”C, some 7°C below the normal L,/H,[ phase transition temperature [4]. The sample was contained in a 1 mm diameter quartz X-ray capillary and was exposed to a monochromatic X-ray beam with an incident flux of 2 x 1010 photons/s at 0.9lOA (13.62 keV) down a 0.3 mm diameter collimator. During the continuous exposure, a series of 2.5 min sequential static X-ray diffraction patterns were recorded on image plates and X-ray sensitive film using a remotely controlled and shuttered translating camera. Quantitative structural characterization of the decaying L, and nascent HII phases was carried out by analyzing the low-angle diffraction peak profiles. The integrated diftiacted intensity associated with the two phases and how this changes with accumulated dose was determined. Under these conditions, integrated intensity is proportional to the amount of lipid in a given phase. The time course for changes in the relative amounts of the two phases was markedly different. The L, phase decayed monotonically during the first 20min of the exposure and then reached a stable final value after 35 min in the X-ray beam. In contrast, there was an initial 1Omin lag in the development of the HII phase, after which it grew rapidly and then stabilized at about the same time that the L, phase ceased to develop. Thin layer chromatographic analysis of radiation-damaged DHPE showed the generation of breakdown products, one of which was identified tentatively as a long chain alcohol, most likely hexadecanol. It is possible, therefore, that in the X-ray beam, DHPE is broken down to yield products which serve to completely destroy the lamellar phase in a concentration-dependent manner. In the early stages of the exposure, however, these products are not present in high enough concentration to effect full expression of a hexagonal lattice. Continued exposure generates more ‘damage product’, which accumulates and eventually nucleates and fuels the production of the HII phase at the expense of the L, phase. The leveling off in the decay of the L, phase and the development of the HII phase at longer exposures to X-rays may reflect a self-limiting process or a steady-state condition in which damage products diffuse away from the irradiated region (a spot 0.3 mm in diameter) into the undamaged L, phase, while fresh material moves into the site of exposure. As the relevant multicomponent
and Chenrr
temperature-composition phase diagram for the system at hand is not known and is likely to be complex, such explanations must remain speculative for now. This dramatic result shows for the first time gross structural rearrangements that have been induced by X-rays in hydrated phosphatidylethanolamine (PE). The consequence of the X-ray exposure was a drastic decrease in temperature of the L,/HII phase transformation boundary and the emergence of a nonlamellar phase in a lamellar environment. The results highlight the need for caution when using bright x-ray sources such as synchrotron radiation. In a separate but related study, we showed that X-radiation damage is free-radical mediated (A Cheng, M Cafiey, abstract in Biophys J 1991, 59:A634). As free radicals have been implicated in cellular senescence, this demonstration that X-radiation damage can effect lamellarjnon-lamellar transformations suggests a means by which free radicals accumulating in cells during aging might compromise membrane integrity and contribute to cell death.
Temperature-induced
transitions
Transition
phase
from the ripple
The temperature driven ripple (Pbt)+L, phase transition in fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) occurs at ~42°C. The kinetics of the transition was examined in a laser temperature (T)jump experiment by making TRXRD measurements simultaneously in the low- and wide-angle region [5]. This allows for the continuous monitoring together of changes in the long- and short-range order that accompany the transition. The infrared erbium glass laser used in the study provided a T-jump rate of 2500 “C s-1, effecting a 5°C rise in a period of 2 111s. Diffraction data were collected with a time resolution of 5111s. To provide difiaction data with a satisfactory signal to noise ratio, statistics had to be built up by repeating the T-jump analysis 100 times. In this study, it is the metastable [6,7] rather than the stable PB phase that was examined. The data show that the phase change is over within 5 ms and that the long- and short-range structural rearrangements that accompany the phase change happen in parallel. In this study [5], the transition in the low-angle diffraction region was characterized by a continuous change in diffraction without any evidence for a distinct coexistence of phases during the transformation. Rapp et al. suggest that the trigger used was sufficiently fast and the degree
Fig. I. Schematic view of the various mesophases, crystalline forms, and states of aggregation adopted by membrane lipids. (a) Lamellar subgel phase (L,); (b) lamellar gel phase (untilted chains, Lb); (c) lamellar gel phase (tilted chains, Lg.); (d) ripple phase (Pv); (e) cross-sectional view of the hydrocarbon chains in a hexagonal close-packed arrangement (view is down the long axis of the chains); (0 lamellar liquid crystal phase (L,); (g) normal hexagonal phase (HI); (h) lamellar liquid crystal phase (L,); (i) inverted hexagonal phase (HI,); (j) inverted cubic phase (space group Pn3m, number 224); (k) inverted cubic phase (space group la3d, number 230); (I) inverted cubic phase (space group lm3m, number 229); (m) tubule; (n) unilamellar vesicle; (0) multilamellar vesicle. The labeled elements refer to parameters or dimensions commonly used in identifying structural features of phases. Adapted from [16*,21-241 with permission.
551
552
lipids
of superheating (relative to the transition temperature) sufficiently large for a f&t martensitic transformation which does not require material diffusion to explain the result. A detailed mechanism, however, was not presented.
Lamellar gel-wbgel
transition
Early studies of the Lg+lamellar subgel (L,) transition in fully hydrated DPPC suggested a new but ill defined mechanism variously referred to as involving ‘sequential relaxation’ [6] and ‘lenient cooperativity’ [8]. The transition was induced by lowering temperature to between -4°C and 4°C and incubating the sample in this temperature range for anywhere from hours to months. This protocol gave rise to complex Lp+L, transition kinetics and to a signature behavior in both low- and wide-angle X-ray diffraction studies, involving an apparent continuous change in characteristic d-spacings from one phase to the other, The enigmatic nature of this behavior was resolved when a protocol was implemented that induced the formation of a small number of subgel-phase nuclei by incubating the sample for a short period at low temperature, followed by a higher temperature incubation in which growth of large subgel domains from a marginally undercooled gel phase could occur [9”]. This behavior follows the classical nucleation-growth hypothesis, which requires phase coexistence during the transformation process. The low- and wide-angle diffraction data obtained with the system close to equilibrium [9**] are entirely consistent with this hypothesis. A remarkable sensitivity of the subgel formation kinetics to X-radiation damage was noted in the study [9**].
Transitions involving chain interdigitated phases
When dispersed in 1 M potassium thiocyanide, DPPC has been shown by X-ray difh-action to form, at low temperature, a lamellar subgel phase with interdigitated chains [lo]. Heating gave rise to a lamellar gel phase which again was characterized by chain interdigitation. This transition has been interpreted [lo] as being of the second-order type because of a continuous change in low- and wide-angle diffraction by DPPC in going from one phase to the other. Calorimetry, however, reveals a peak as opposed to a slope change in the heat capacity versus temperature plot in the vicinity of the phase change detected by diffraction. This result is at variance with the proposed second-order transition. Upon continued heating, the interdigitated lamellar gel phase transforms to the Lo phase. Phase coexistence during the course of the dynamic transformation is consistent with this high-temperature transition being first order.
Cubic phase transitions
The temperature-composition phase diagram of monomyristolein (a monoacylglycerol with a 14-carbon cir-monounsaturated acyl chain) in water includes the lamellar crystalline phase, the Lo phase, the fluid isotropic (FI) ph ase, and two inverted cubic phases (Pn3m and Ia3d; Fig. lj,k; Fig. 2). The cubic phases are notoriously inclined to undercool [l l-131. Such metastability can complicate the construction of a true equilibrium phase diagram. On the other hand, Mariani et al. [13] have proposed that the metastable character of cubic phases is exploited by certain thermophilic Archaebmteria as a means for adapting to the rather harsh environments in which the organisms live.
In an effort to understand the phenomenon, cubic phase undercooling in monomyristolein has been examined in some detail [14**]. To this end, the temperature of samples in the cubic phase was adjusted to a value where the L, phase represents equilibrium behavior. Cooling-induced structure and phase changes were monitored continuously by recording low-angle difl%action patterns from the samples using a streak camera (Fig. 2a). Interestingly, the cubicjlamellar transition rate decreased with increasing sample hydration. Furthermore, there was an initial rapid increase in cubic phase unit cell size that was accompanied by the formation of a significant amount of the L, phase. The following mechanism accounts for such behavior. At any given temperature, there is a unit cell size that optimally stabilizes the undercooled cubic phase. Furthermore, as this unit cell size is approached, the lifetime of the metastable state lengthens. Undercooling the cubic phase results in a dramatic increase in the size of the cubic unit cell due mainly to water imbibition. If, upon cooling, the cubic phase cannot access the desired lattice size because of a limited supply of available water, the sample begins to convert from the undercooled cubic phase to the equilibrium Lo phase. If, on the other hand, water is immediately available (or can be made available) to the metastable cubic phase, the desired lattice parameter can be approached.
According to the monomyristolein phase diagram (Fig. 2c), the cubicjlamellar phase transition is a water releasing event at overall sample compositions of greater than 32% (w/w) water. The water liberated upon the partial transformation of an undercooled cubic phase to the Lo phase can be taken up by any untransformed cubic phase, allowing it to swell. Depending on sample composition, different amounts of the L, phase will be required to form in order that the necessary water can be made available to the undercooled cubic phase. Consistent with this model, it was found that cubic-phase swelling indeed accompanies the formation of the Lo phase in all but the most hydrated sample.
State University,
Columbus,
In the past two years, the kinetics of transitions
involving
USA
the assorted lamellar
and inverted hexagonal and cubic phases in bulk hydrated lipid systems have been established
using a variety of physical techniques.
kinetic data have lead to a transition
Current
Opinion
mechanism
in Structural
Biology
Introduction
In several cases, the
being deciphered.
1995,
5:548-555
learn why nature endows biomembranes enormous lipid-type diversity.
In the context of the living cell, the lipid bilayer can be viewed as recruiting proteins and other molecules for the purpose of generating a selectively permeable, signal transducing supramolecular aggregate known as the biological membrane. The lipid component of membranes, and indeed of other lipid-rich bioaggregates, has the inherent capacity to adopt a variety of so-called liquid crystalline states, or mesophases, more ordered than the liquid but less so than the crystalline solid (Fig. 1). The lipid bilayer represents one such state, referred to as the lamellar or smectic phase. The remainder are grouped together in the so-called non-bilayer phase category. The particular liquid crystal phase accessed by manipulating the conditions depends on temperature, pressure, lipid concentration and composition of the aqueous dispersing medium. Interestingly, the majority of isolated membrane lipids are content to form one or other of the non-bilayer phases at or close to physiological conditions. In the course of undergoing such bilayer-+non-bilayer phase changes, intermediates, reminiscent of structures proposed to appear transiently during the ubiquitous process of membrane fusion, are likely to develop. This accounts, in part, for the interest in lipid-based phase-transition kinetics and mechanism studies. One way of deciphering mechanism is to establish the kinetic parameters of the transition. It is such on-going endeavors that will be covered in this review. A long-term objective in this area is to establish how lipid molecular structure and composition in mixed lipid systems affect phase-transition kinetics and mechanism. Such insights will provide for rational design strategies aimed at effecting control over not only membrane structure and function in intact organisms, but also over performance characteristics in reconstituted and formulated systems. Along the way, we hope to
with such an
To consider the area of lipid phase transition kinetics a burgeoning field is likely to be an overstatement of the case: probably 10 to 20 groups worldwide are active in the area. This pales in comparison with other areas within the realm of structural biology, although the numbers make a little more sense when viewed in the context of the complexity of the systems being studied, the sophisticated measurements needed to extract useful information and the techniques currently available for their investigation. This review, which amounts to a snapshot in time of what is happening in the field, gives a flavor of the enormous variety that exists in the systems actively under investigation and the techniques being used for the purpose. The review is of limited scope in that it covers only the period from January 1993 onwards and the literature dealing with experimental aspects of relatively fast phase transitions in bulk hydrated lipid systems. Because of the narrow focus, it has been possible to include in the review all relevant published work that has come to our attention.
Pressure-induced
transitions
Transitions
the lamellar
involving
phase using
pressure jump
The kinetics and mechanism of the barotropic (pressuresensitive) lamellar gel (Lbr)+lamellar liquid crystal (L,) phase transition in fully hydrated 1,2-dihexadecylsn-glycero-3-phosphoethanolamine (DHPE) has been studied using time-resolved X-ray diffraction (TRXRD) [l”]. The phase transition was induced by pressure jumps of varying amplitudes in the pressurization and
Abbreviations DHPE-l,2-dihexadecyl-sn-glycero-3-phosphoethanolamine; tit,-inverted
hexagonal
L,-subgel
L,-lamellar
phase; MLV-multilamellar Pn3m
548
phase;
and la3drubic
liquid
DPPC-l,2-dipalmitoyl-sn-glycero-3-phosphocholine;
crystalline
vesicle;
P-pressure;
space groups;
0 Current
phase;
Lg-lamellar
l-temperature;
Biology
gel phase;
PC-phosphatidylcholine; TRXRD-time-resolved
Ltd ISSN 0959-440X
L~S-lamellar
gel phase with
PE-phosphatidylethanolamine; X-ray diffraction.
tilted
chains:
Kinetics of lipid phase changes Caffrey and Cheng
depressurization directions at controlled temperature dieacted X-rays (78°C). Both 1ow- and wide-angle were recorded simultaneously in real-time which allowed for the direct and quantitative characterization of the long-range (lamellar repeat spacing) and short-range (chain packing) structural order of DHPE during a kinetic experiment. The image-processed live-time X-ray diffraction data were fitted using a non-linear least squares model and the parameters of the fits were monitored continuously throughout the transition. The barotropic transitions from the L, to the LB’ phase phase were two-state and from the LB’ to the L, (no formation of intermediates apparent during the transition) to within the sensitivity limits of the method. The corresponding transit time (the time during which both phases coexist) associated with the long- and shortrange order of the pressurization-induced L,+Lp phase transition decreased to a limiting value of approximately 50 ms with increasing pressure-jump amplitude. This limiting value was close to the response time of the detector/recording system. Thus, the intrinsic transit time of this transition in fully hydrated DHPE at 78°C was less than or equal to 50 ms. In contrast, the depressurizationinduced Lbt+L, phase transition was slower, taking approximately 1 s to complete, and occurred with no obvious dependence of the transit time on pressure-jump amplitude. In the depressurization-jump experiment, the lipid responded rapidly to the pressure jump in the LB phase up to the rate-determining Lp+L, transition, by converting to a low-pressure form of the LB’ phase. The latter, enigmatic behavior, suggestive of a transition intermediate, is wont to complicate the interpretation of phase transition kinetic measurements and is worthy of note by researchers in the area. Pressure jump has also been used to study the kinetics of the lamellar/inverted hexagonal (HI,; Fig. li) phase in lipids using TRXRD with a resolution of 9 ms (M Kreichbaum et al., Biopllys J 1993, 64:A296). With pressure-jump amplitudes in excess of 1 kbar the transition was completed within the time resolution of the measurement. A slower transition was observed with smaller amplitude jumps.
Lamellar
gel/liquid
crystal phase transition
in binary
lipid
mixtures
A powerful stationary relaxation technique employing volume perturbation calorimetry has been used by Biltonen and Ye ([2]; see also references therein for seminal work in this area) to study the kinetics and mechanism of the chain order/disorder transition in binary (two component) lipid membranes. In this technique, the sample housed under essentially adiabatic conditions is subjected to a small periodic change in volume by means of a piezoelectric crystal stack. The pressure and temperature response of the sample is monitored continuously during the course of the measurement performed over several decades of perturbation frequencies. The impulse-response data are analyzed in the frequency
domain, from which can be obtained the relaxation rate and insights into transition mechanism. When applied to relatively simple binary phosphatidylcholine (PC) bilayer mixtures undergoing the chain order/disorder transition, four results were found. First, the relaxation process is considerably more complex than that observed with single component systems and consists of more than a single exponential decay. Second, the relaxation rate for the mixture is at least an order of magnitude higher than that for single component membranes. Third, the relaxation rate enhancement was pronounced when the minor component of the lipid mixture had the higher temperature for the order/disorder transition. Fourth, the relaxation time versus temperature profile had two maxima regardless of whether the corresponding equilibrium heat capacity function had one or two peaks. These results suggest either that such mixed-lipid membranes exhibit a form of dynamic phase separation in the bilayer plane and/or that molecular diffusion plays an important function in defining the kinetics of in-plane phase separation.
Lamellar
gel/liquid
single component
crystal partial
phase transition
in a
system
In order to extend the volume perturbation calorimetry approach described above, an instrument has been built that allows for an analogous perturbation to be imposed on the sample with the added feature of being able to monitor structure changes directly by means of simultaneously performed TRXRD. Accordingly, we have used synchrotron-based TRXRD to monitor both low- and wide-angle difhaction changes during a partial L,/lamellar gel (LB) mesophase transition in fully hydrated monoelaidin (a monoacylglycerol with an 18-carbon nuns-unsaturated acyl chain) multilamellar vesicles in response to small pressure oscillations applied over four decades in frequency (A Cheng, M Caffrey, abstract in Biophys J 1995, 68A96). The sinusoidal pressure oscillation amplitude was small enough to elicit a linear response. The response amplitude and phase shift were both determined as a function of perturbation frequency. The fractional phase conversion response spectra for the two structure changes monitored (mesophase lamellarity and chain conformation) were very similar, indicating that the chain order/disorder transition correlates well with the lamellar repeat spacing change. The data are consistent with a transition best described by the Kolmogorov-Avrami kinetic model [3], having an effective dimensionality close to unity (0.78 f 0.05 and 0.9 kO.1 for the low- and wide-angle data, respectively) and a structural relaxation time of 13 f 2 and 10 f 3 s for the low- and wide-angle data, respectively. We have interpreted the results in the context of a bidirectional layer-by-layer transition mechanism for the La/L8 phase change. Although the transition is predominantly interface controlled, material diffusion in the form of water flux is likely to play a modest role in the phase interconversion.
549
550
lipids
(B) lamellar gel (LB)
(A)sub-gel0-d
, ripple wavelength
(E) chain packing
(C) lamellar gel-tilted (Lv)
(F) lamellar liquid crystal (L,)
d,=d,,=a.cos
(G) normal hexagonal
d hex
(HI)
(H) lamellar liquid crystal (L,)
(1)inverted hexagonal (HII)
T
T
dhex=do,=
d her
3, 2
w
(J) Cubic Pn3m (Q224)
(M) tubule
(K) Cubic la3d (Q230)
(N) unilamellar vesicle
(L) Cubic lm3m (Q22g)
(0) multi-lamellar
vesicle
Kinetics of lipid phase changes Caffrey
An X-ray-induced
transition:
lamellar/hexagonal
phase transition The X-radiation induced partial L, +HII phase transformation was observed in a fully hydrated DHPE sample at 79”C, some 7°C below the normal L,/H,[ phase transition temperature [4]. The sample was contained in a 1 mm diameter quartz X-ray capillary and was exposed to a monochromatic X-ray beam with an incident flux of 2 x 1010 photons/s at 0.9lOA (13.62 keV) down a 0.3 mm diameter collimator. During the continuous exposure, a series of 2.5 min sequential static X-ray diffraction patterns were recorded on image plates and X-ray sensitive film using a remotely controlled and shuttered translating camera. Quantitative structural characterization of the decaying L, and nascent HII phases was carried out by analyzing the low-angle diffraction peak profiles. The integrated diftiacted intensity associated with the two phases and how this changes with accumulated dose was determined. Under these conditions, integrated intensity is proportional to the amount of lipid in a given phase. The time course for changes in the relative amounts of the two phases was markedly different. The L, phase decayed monotonically during the first 20min of the exposure and then reached a stable final value after 35 min in the X-ray beam. In contrast, there was an initial 1Omin lag in the development of the HII phase, after which it grew rapidly and then stabilized at about the same time that the L, phase ceased to develop. Thin layer chromatographic analysis of radiation-damaged DHPE showed the generation of breakdown products, one of which was identified tentatively as a long chain alcohol, most likely hexadecanol. It is possible, therefore, that in the X-ray beam, DHPE is broken down to yield products which serve to completely destroy the lamellar phase in a concentration-dependent manner. In the early stages of the exposure, however, these products are not present in high enough concentration to effect full expression of a hexagonal lattice. Continued exposure generates more ‘damage product’, which accumulates and eventually nucleates and fuels the production of the HII phase at the expense of the L, phase. The leveling off in the decay of the L, phase and the development of the HII phase at longer exposures to X-rays may reflect a self-limiting process or a steady-state condition in which damage products diffuse away from the irradiated region (a spot 0.3 mm in diameter) into the undamaged L, phase, while fresh material moves into the site of exposure. As the relevant multicomponent
and Chenrr
temperature-composition phase diagram for the system at hand is not known and is likely to be complex, such explanations must remain speculative for now. This dramatic result shows for the first time gross structural rearrangements that have been induced by X-rays in hydrated phosphatidylethanolamine (PE). The consequence of the X-ray exposure was a drastic decrease in temperature of the L,/HII phase transformation boundary and the emergence of a nonlamellar phase in a lamellar environment. The results highlight the need for caution when using bright x-ray sources such as synchrotron radiation. In a separate but related study, we showed that X-radiation damage is free-radical mediated (A Cheng, M Cafiey, abstract in Biophys J 1991, 59:A634). As free radicals have been implicated in cellular senescence, this demonstration that X-radiation damage can effect lamellarjnon-lamellar transformations suggests a means by which free radicals accumulating in cells during aging might compromise membrane integrity and contribute to cell death.
Temperature-induced
transitions
Transition
phase
from the ripple
The temperature driven ripple (Pbt)+L, phase transition in fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) occurs at ~42°C. The kinetics of the transition was examined in a laser temperature (T)jump experiment by making TRXRD measurements simultaneously in the low- and wide-angle region [5]. This allows for the continuous monitoring together of changes in the long- and short-range order that accompany the transition. The infrared erbium glass laser used in the study provided a T-jump rate of 2500 “C s-1, effecting a 5°C rise in a period of 2 111s. Diffraction data were collected with a time resolution of 5111s. To provide difiaction data with a satisfactory signal to noise ratio, statistics had to be built up by repeating the T-jump analysis 100 times. In this study, it is the metastable [6,7] rather than the stable PB phase that was examined. The data show that the phase change is over within 5 ms and that the long- and short-range structural rearrangements that accompany the phase change happen in parallel. In this study [5], the transition in the low-angle diffraction region was characterized by a continuous change in diffraction without any evidence for a distinct coexistence of phases during the transformation. Rapp et al. suggest that the trigger used was sufficiently fast and the degree
Fig. I. Schematic view of the various mesophases, crystalline forms, and states of aggregation adopted by membrane lipids. (a) Lamellar subgel phase (L,); (b) lamellar gel phase (untilted chains, Lb); (c) lamellar gel phase (tilted chains, Lg.); (d) ripple phase (Pv); (e) cross-sectional view of the hydrocarbon chains in a hexagonal close-packed arrangement (view is down the long axis of the chains); (0 lamellar liquid crystal phase (L,); (g) normal hexagonal phase (HI); (h) lamellar liquid crystal phase (L,); (i) inverted hexagonal phase (HI,); (j) inverted cubic phase (space group Pn3m, number 224); (k) inverted cubic phase (space group la3d, number 230); (I) inverted cubic phase (space group lm3m, number 229); (m) tubule; (n) unilamellar vesicle; (0) multilamellar vesicle. The labeled elements refer to parameters or dimensions commonly used in identifying structural features of phases. Adapted from [16*,21-241 with permission.
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lipids
of superheating (relative to the transition temperature) sufficiently large for a f&t martensitic transformation which does not require material diffusion to explain the result. A detailed mechanism, however, was not presented.
Lamellar gel-wbgel
transition
Early studies of the Lg+lamellar subgel (L,) transition in fully hydrated DPPC suggested a new but ill defined mechanism variously referred to as involving ‘sequential relaxation’ [6] and ‘lenient cooperativity’ [8]. The transition was induced by lowering temperature to between -4°C and 4°C and incubating the sample in this temperature range for anywhere from hours to months. This protocol gave rise to complex Lp+L, transition kinetics and to a signature behavior in both low- and wide-angle X-ray diffraction studies, involving an apparent continuous change in characteristic d-spacings from one phase to the other, The enigmatic nature of this behavior was resolved when a protocol was implemented that induced the formation of a small number of subgel-phase nuclei by incubating the sample for a short period at low temperature, followed by a higher temperature incubation in which growth of large subgel domains from a marginally undercooled gel phase could occur [9”]. This behavior follows the classical nucleation-growth hypothesis, which requires phase coexistence during the transformation process. The low- and wide-angle diffraction data obtained with the system close to equilibrium [9**] are entirely consistent with this hypothesis. A remarkable sensitivity of the subgel formation kinetics to X-radiation damage was noted in the study [9**].
Transitions involving chain interdigitated phases
When dispersed in 1 M potassium thiocyanide, DPPC has been shown by X-ray difh-action to form, at low temperature, a lamellar subgel phase with interdigitated chains [lo]. Heating gave rise to a lamellar gel phase which again was characterized by chain interdigitation. This transition has been interpreted [lo] as being of the second-order type because of a continuous change in low- and wide-angle diffraction by DPPC in going from one phase to the other. Calorimetry, however, reveals a peak as opposed to a slope change in the heat capacity versus temperature plot in the vicinity of the phase change detected by diffraction. This result is at variance with the proposed second-order transition. Upon continued heating, the interdigitated lamellar gel phase transforms to the Lo phase. Phase coexistence during the course of the dynamic transformation is consistent with this high-temperature transition being first order.
Cubic phase transitions
The temperature-composition phase diagram of monomyristolein (a monoacylglycerol with a 14-carbon cir-monounsaturated acyl chain) in water includes the lamellar crystalline phase, the Lo phase, the fluid isotropic (FI) ph ase, and two inverted cubic phases (Pn3m and Ia3d; Fig. lj,k; Fig. 2). The cubic phases are notoriously inclined to undercool [l l-131. Such metastability can complicate the construction of a true equilibrium phase diagram. On the other hand, Mariani et al. [13] have proposed that the metastable character of cubic phases is exploited by certain thermophilic Archaebmteria as a means for adapting to the rather harsh environments in which the organisms live.
In an effort to understand the phenomenon, cubic phase undercooling in monomyristolein has been examined in some detail [14**]. To this end, the temperature of samples in the cubic phase was adjusted to a value where the L, phase represents equilibrium behavior. Cooling-induced structure and phase changes were monitored continuously by recording low-angle difl%action patterns from the samples using a streak camera (Fig. 2a). Interestingly, the cubicjlamellar transition rate decreased with increasing sample hydration. Furthermore, there was an initial rapid increase in cubic phase unit cell size that was accompanied by the formation of a significant amount of the L, phase. The following mechanism accounts for such behavior. At any given temperature, there is a unit cell size that optimally stabilizes the undercooled cubic phase. Furthermore, as this unit cell size is approached, the lifetime of the metastable state lengthens. Undercooling the cubic phase results in a dramatic increase in the size of the cubic unit cell due mainly to water imbibition. If, upon cooling, the cubic phase cannot access the desired lattice size because of a limited supply of available water, the sample begins to convert from the undercooled cubic phase to the equilibrium Lo phase. If, on the other hand, water is immediately available (or can be made available) to the metastable cubic phase, the desired lattice parameter can be approached.
According to the monomyristolein phase diagram (Fig. 2c), the cubicjlamellar phase transition is a water releasing event at overall sample compositions of greater than 32% (w/w) water. The water liberated upon the partial transformation of an undercooled cubic phase to the Lo phase can be taken up by any untransformed cubic phase, allowing it to swell. Depending on sample composition, different amounts of the L, phase will be required to form in order that the necessary water can be made available to the undercooled cubic phase. Consistent with this model, it was found that cubic-phase swelling indeed accompanies the formation of the Lo phase in all but the most hydrated sample.