© Copyright 2001 by the American Chemical Society
VOLUME 105, NUMBER 14, APRIL 12, 2001
LETTERS Observation of Liquid-Crystal-Like Ferroelectric Behavior in a Biological Membrane Irina Ermolina, Alina Strinkovski, Aaron Lewis, and Yuri Feldman* The Department of Applied Physics, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel ReceiVed: March 21, 2000; In Final Form: NoVember 21, 2000
In this contribution, we report the application of time domain dielectric spectroscopy from 500 kHz to 1 GHz and differential scanning calorimetry to bacteriorhodopsin containing purple membrane films. The results of these measurements unexpectedly show that the oriented purple membrane has a unique liquid-crystal-like ferroelectric behavior. The dielectric behavior can be considered as soft mode relaxation processes in ferroelectric liquid crystals near smectic-C*-smectic-A phase transition. Such a phenomenon has not been previously observed in biological systems.
Bacteriorhodopsin (bR) is an integral membrane protein that is found in the purple membrane of the bacterium Halobacterium salinarium. The function of bR is that of a light driven proton pump. In the past 25 years since its discovery, numerous biophysical measurements have been performed on this important system in biological energy transduction.1-4 However, the fundamental mechanisms that underlie this molecular system remain unsolved. Nonetheless, it is thought that a key component in the function of this membrane protein is the movement of charge and the motion of dipolar groups in the membrane.3 A well-known methodology to investigate such movements in charged and polar groups is dielectric spectroscopy. Despite this, such a technology has not yet been applied to the bR system. In this contribution, we report the application of time domain dielectric spectroscopy (TDDS) from 500 kHz to 1 GHz to bR containing purple membranes. The purple membrane film was produced by the normal procedure.5 With these membranes two groups of samples were prepared as oriented and nonoriented films. The oriented sample (the film of 30 µm thickness) was prepared by depositing purple membrane fragments on the electrode in an electric field. All samples were investigated using TDDS as a function of * To whom correspondence should be addressed. E-mail: yurif@ vms.huji.ac.il.
temperature and by differential scanning calorimetry (DSC). The temperature interval that was investigated was between 5 and 27 °C, and measurements were recorded as a result of both cooling and heating the sample. All of the measurements were done at room atmosphere and a humidity of around 60%. In addition, the TDDS measurements were also performed with and without the influence of an external electric field at 23 °C. Two different time domain dielectric spectrometers were used for the bR dielectric measurements. The temperature measurements in a broad frequency range (500 kHz-1 GHz) were made using a TDS-2.1 spectrometer (Dipole TDS Ltd., Jerusalem).6 The influence of the external electric field on the dielectric properties of a bR dry film in the frequency range of 50 MHz10 GHz were made using a HP digitizing sampling oscilloscope HP54120B. The external electric filed was applied using a Matrix AX322 power supply connected to a HP pulse generator and a sampling oscilloscope. The external electric field is applied through the bias tee (Picosecond Pulse Labs, 5530A).7 In the case of both systems, the sample cell was arranged in the form of a lumped capacitance terminating the coaxial line. bR thin films were placed on the electrodes of the lumped capacitance cell. In TDDS measurements, a fast rising (∼20 ps) step functionlike electrical pulse lasting over a time period of 10 µs is applied
10.1021/jp001054y CCC: $20.00 © 2001 American Chemical Society Published on Web 03/16/2001
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Letters
Figure 1. Dipole correlation function Γ(t) of bR at 23 °C, provided by TDDS. (O) is the experimental data; the solid line corresponds to a fit by the sum of the Debye relaxation processes, and the dashed line shows the low-frequency process, with the effective amplitude being equal to 0.26 and the correlation time being equal to 1158 ns.
Figure 2. Temperature dependence of the dielectric permittivity of the bR membrane in the low-frequency limit of TDDS measurements; (2) denotes heating of sample; (O) denotes cooling of sample. The measurement accuracy of the dielectric permittivity was better than 3%.6
to a sample that is sandwiched between two electrodes. The reflected electrical signal, after a Fourier transform, can be analyzed in the frequency domain to probe the spectrum of the complex dielectric permittivity of the sample, revealing the polarization components as the dielectric permittivity and losses of the sample. These measurements probe the vector of polarization that is related to the orientational mobility of a variety of dipole groups that occur in biological systems. This can also be analyzed simultaneously in the time domain in terms of macroscopic dipole fluctuations in the system. In the time domain, the result is represented as a macroscopic dipole correlation function Γ(t) (DCF):
Γ(t) )
〈M h (0)‚M h (t)〉 〈M h (0)‚M h (0)〉
(1)
where M(t) is the macroscopic fluctuation dipole moment of the sample volume unit, which is equal to the vector sum of all of the molecular dipoles; the symbol 〈 〉 denotes averaging of the ensemble. The laws governing the decay function Γ(t) and its rate of decay are directly related to the structural and kinetic properties of the sample and characterize the macroscopic properties of the system studied. The general principles of TDDS and a detailed description of its instrumentation and software algorithms have been described elsewhere.6 The DCF observed in TDDS measurements have complex, nonexponential behavior (Figure 1) and in the 1st approximation may be represented as a sum of exponential processes. In this paper, we only analyze and discuss the long relaxation process. In future papers, we will focus on all of the scales of molecular motion that are represented in the correlation function. The temperature dependence of the static dielectric permittivity during the complex protocol of a bR cooling-heating cycle is presented in Figure 2. A crossover of the heating curve with the cooling curve is observed at 18 °C. About the same temperature was manifested in DSC measurements where the oriented bR membrane showed an endothermic process (Figure 3). The TDDS measurements with application of an external electric field exhibit a strong dependence of dielectric strength both on the amplitude and the direction of the field (Figure 4). The main common feature of the observed results is the presence of some transition stimulated both by temperature and an external electric field.
Figure 3. Heat flow dependence for the oriented and nonoriented bR membrane.
Figure 4. dc-bias field dependence of the dielectric strength at 25 °C. The external field changed according to the following route (see arrows): from 0 to +35 Kv/cm (4); from +35 to -35 Kv/cm (9); from -35 to +35 Kv/cm (x). The accuracy of the dielectric strength evaluation was better than 15%.
The first question that we should address is the nature of the thermal transition that is observed for this stack of purple membranes. From one point of view, the temperature and the nature of the transitions are reminiscent of phase transitions of
Letters lipids that have been studied for many years. Nonetheless, this thermal transition is practically nondetectable in the case where the membranes were essentially nonoriented. This difference between oriented and nonoriented membranes leads us to conclude that the situation in this stack of purple membranes is more complex than that which is simply observed in a standard lipid phase transition. In the case of this stack of purple membranes, the protein molecules in each membrane fragment exhibit excursions on both the cytoplasmic and extra cellular sides of the membrane. These regions of excursion in the protein chain are in all probability interacting with each other in a defined way in the oriented purple membrane stack. These interactions present barriers to the rotation of bR molecules, and these barriers can only be crossed with certain thermal properties of lipids. At a specific temperature, which is evident in the differential calorimetry and is about 18 °C, we see a transition that could be ascribed to the freezing of the lipid chains. However, if it was not for the interacting protein chains, we suggest that we would not be able to detect this transition. A clue to this deduction comes from the results on the nonoriented purple membrane in which only the very hint of a transition is seen. Thus, the freezing of the lipids is not enough to observe this thermal transition. A scenario that could explain this observation would be via an indirect affect of the alteration of the lipid chain conformation on the protein libration. This could allow conformational freedom for the interacting protein molecules. Thus, in view of the nature of the thermal transition, it would seem that the lipid chain conformation is a critical component of this librational motion. The presence of the protein interactions however provides for the cooperative nature of the transition between the cytoplasmic and extra cellular regions of the protein chain. The results obtained can now be reinterpreted within this suggested view for this stack of purple membranes. Namely, the difference in the differential calorimetry between the oriented and the nonoriented membranes can be understood as a result of the conformational alteration in the lipid chains permitting the libration of protein molecules. This libration then occurs in a cooperative fashion as a result of protein-protein interactions. Thus, in the nonoriented membranes the interactions which are relatively more random than in the oriented case show only a very slight affect from the injection of heat. It is also very likely that there is some residual orientation even in the essentially nonoriented case, and this could be the cause of the small effect that has been observed. Now, let us consider the dielectric spectroscopy, which also observes a similar transition occurring in the same range of temperatures as that of the differential calorimetry. The dielectric spectroscopy is measuring the vector of polarization related to the orientation mobility of protein dipoles. As a result of the postulated librational motion of the protein molecules discussed above there would be an alteration of the dipole that is associated with the uneven excursions of the helical protein through the membrane. This results in a gross alteration in the dipolar characteristics as libration occurs. Furthermore, the cooperative nature of this dielectric transition in oriented membranes further supports the view that the origin of the cooperation is the protein-protein interactions that cooperate in the librational motion. In addition, the measurements with electric fields add further support to this notion. If the electric fields are high enough, then even at lower temperatures the librational motion could be induced by the fact that the dipole vector of the uneven tans membrane protein excursions is at some angle to the normal
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Figure 5. Frequency of maximum loss for the oriented (9) and nonoriented (O) bR membrane film vs temperature.
to the membrane. Thus, all our results indicate that the origin of the cooperative transitions that we have observed arises from protein-protein interactions. These transitions can be detected either by causing a heat-induced alteration in the lipid chain conformation or by imposed electric fields that could induce protein librational motions at even lower temperatures. To understand the physical nature of the transition presented above, we note that it is well-known that the bR macromolecule has a significant dipole moment (50-100 D).8 This dipole is geometrically organized in the purple membrane, which has three levels of structural organization. The first one is the macromolecular assembly of the bR molecules in the purple membrane as triads.9 The second level consists of the planar hexagonal structure of the protein triads9 in this membrane, and finally, there is a third level of organization of this planar ordering of membrane proteins as an interacting oriented multilayered structure of membrane fragments. This specific three-level structure with a dominant dipole moment oriented in the bR protein seems to have unique ferroelectric liquidcrystal properties. The proof of this supposition can be found in the temperature and electric-field dependencies of the dielectric parameters and the properties of heat flow vs temperature to be discussed below. The ferroelectric nature of some materials is based upon a body of evidence that is very well-known. First, there is the validity of the Curie-Weiss law.10 Second, there is the temperature and dc-bias hysteresis of the dielectric strength.10,11 Third, there is the appearance of the DSC peak near the smecticA- smectic-C* phase transition.12 An analysis of our results shows that there is in our data a specific behavior for the relaxation frequency with a well-pronounced minimum near a temperature of 17-18 °C (Figure 5). This temperature interval is similar to what is seen in the DSC peak (Figure 3) for the oriented membrane. Furthermore, by plotting the dielectric strength vs temperature (Figure 6) a dependence was obtained which agrees with the crossover behavior of 1/∆ that was calculated for a soft mode relaxation processes in ferro-electric liquid crystals near smectic-C*-smectic-A phase transition.12-14 These theoretical predictions were obtained for a system with a strong biquadratic coupling between tilt and polarization. The dipole that is seen in bR is due to the unequal number of helical excursions through the membrane, leaving the protein molecule with a net dipole. Each of these helices are at a different angle relative to the membrane normal, and such a tilt of the net observed dipole would be consistent with a biquatratic coupling between tilt and polarization.12,15
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Letters assistance, and G. Bitton for helpful discussions. I.E. thanks the Valazzi-Pikovsky Fellowship Fund for the support of this work. References and Notes
Figure 6. Temperature dependence of the inverse dielectric strength.
In summary, the purple membrane over the years has exhibited numerous interesting physical properties that have led to new understandings of both membrane structure and membrane organization. This paper has shown that these membrane assemblies also have a unique liquid-crystal-like ferroelectric behavior and it is anticipated that this observation will allow us to further probe the properties of this important membrane protein system. Acknowledgment. We thank D. Golodnitsky for the DSC measurements, T. Skodvin for TDDS high voltage measurement
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