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High pressure synchrotron X-ray diffraction studies of biological molecules using the diamond anvil technique
Nuclear tnstruments and Methods in Physics Research A 368 ( 1996) X47-8.51
NUCLeUI INSTMMENTS &METnooS IN PHYSICS “Y!Z!?”
EISEVIER
High pressure synchrotron X-ray diffraction studies of biological molecules using the diamond anvil technique C. Czeslik”, R. Malessa”, R. Winter”.*, G. Rapph
Received
13
April 1995; revised
form received 2 August 1995
Abstract A system for high pressure synchrotron X-ray diffraction studies of biological samples in a diamond anvil cell (DAC) is described. It is capable of operating in the whole temperature and pressure range of interest for studies of biological molecules, i.e.. in the temperature range from -40 to IOO”C at pressures between 1 bar and 50 kbar. The pressure is calibrated by measuring the pressure dependence of the ruby fluorescence line at 694 nm. Two linear detectors connected in series are used to measure simultaneously the small- and wide-angle X-ray scattering. The advantage of the experimental technique is threefold: Firstly, the amount of sample can be kept to a minimum (ea. 30 nl) using the high intensity of synchrotron radiation. Secondly, only the diamond anvil technique allows to reach extreme pressures. Thirdly, the use of the dual detector system allows recording of diffraction data both in the small- and wide-angle region at the same time. Examples of hitherto unknown phases of aqueous lipid and protein samples illustrate the potential of the system.
1. Intr~uction In recent years considerable effort has been put into studying pressure effects on bio~hemicai systems [l-7]. Besides the physico-chemical interest in using pressure as a thermodynamic variable to understand the structure. phase behaviour and dynamics of biological molecules and to develop and prove theoretical approaches, high pressure is also of considerable physiological and biotechnological interest. For example. pressure studies are of interest to understand the physiology of deep sea organisms (in the deep sea pressures up to 1.2 kbar occur), the sensitivity of excitable cell membranes to pressure (“high pressure nervous syndrom”), and the antagonistic effect of pressure to anesthetic action. In food technology. application of high hydrostatic pressure in food sterilization and processing has been a central issue of research and development in Japan since several years [3]. Due to pressure-induced ice formation. the pressure range for studying biomolecules is mainly restricted to pressures below 20 kbar IS]. Up to this pressure, the main effects of applied pressure derive from changes of intermolecular distances, which generally lead to conformational changes, but also from ionization. aggregation and dissociation of biological molecules. The structure and * Corresponding
author. ‘Tel. 1-49 23 1 755 3900. fax +49
7-3 I
755 3901,
016%9001/9&/$15.00 SSD/
0
0
16%9003( 95
1996 Elsevier Science B.V. All rights reserved )OO78
l-1
stability of biological molecules depend mainly on three kinds of interactions: ionic. hydrophobic and hydrogen bonding. Due to the electrostrictive effect of separated charges, ion pairs in aqueous solution are strongly stabilized by hydrostatic pressure. The pH of water decreases with pressure and the exposure of hydrophobic residues, occuring for instance during unfolding of proteins, is favoured by elevated pressures. Hydrogen bond formation in biomolecules is generally accompanied by a negligibly small positive or negative volume change. Considering the fundamental principles of the solution structure of globular proteins. i.e., optimum packing of the hydrophobic core. minimum hydrophobic surface area, and formation of ion pairs within and between subunits. it is evident that hydrostatjc pressure below 20 kbar mainly affects both the tertiary and quatemary structure of proteins. In the past thermodynamic and spectroscopic methods have been widely used for studies on high pressure phase behaviour and structure of biomolecules. and these studies were restricted to pressures below 3-S kbar (see. e.g.. Refs. 19-181). Only recently. the high-pressure FI’IR technique has been developed for measurements at higher pressures 12,191. X-ray and neutron diffraction work was also limited to about 4 kbar in the past [ 14- 171. The availability of high flux synchrotron X-ray sources and recent advances in X-ray detector development, in conjunction with the diamond anvil technique. allow to extend the pressure range up to extreme pressures. Although the
848
C. Czeslik et al. I Nucl. Instr. and Meth. in Phys. Res. A 368 (1996) 847-851
DAC is already a widely used tool for diffraction work in research of the solid state [20], its potential for studies of biological solution structures at elevated pressures has not been recognized so far. We present a setup for studying simultaneously the small- and wide-angle diffraction pattern of biomolecules and biomolecul~ aggregates at pressures up to 50 kbar, and its performance is illustrated by examples of recent experiments.
2. Experimental setup A diamond anvil cell with type IIa diamonds (Diamond Optics, Tucson, USA) was used for the high pressure X-ray diffraction experiments. The sample under study is positioned between the two diamonds (see Fig. 1) which have a cut with 16 facets. The diameter of the anvil surface is 0.6 mm. The total path length of the X-ray beam through the diamonds is about 5 mm, which leads to a reduction of the primary photon intensity by a factor of about lo4 (at A = 1.5 A). The sample is sealed in a gasket (made of Inconel 718 with 53% Ni. 19% Fe, and 19% Cr) of 0.25 mm thickness with a hole of 0.37 mm diameter. The total volume of the sample is about 30 nl. The diamond anvils are held in a thermostated jacket. Since the sample volume is very small and the absorption of the X-ray intensity by the diamonds is very high, the diffracted intensity is extremely weak and exposure times of several minutes (typically 10 min) are required at common synchro~on X-ray sources. Pressure is calibrated by measuring the shift of the fluorescence line at 694 nm of a small ruby chip which is added to the sample in the gasket hole (dhldp = 0.036 nm/kbar [21,22]).
The small- and wide-angle X-ray diffraction experiments were performed at beamline X13 of the EMBL outstation at DESY in Hamburg. With the germanium monochromator installed, the wavelength of the X-rays is fixed to 1.5 A. Sets of tungsten slits are used to adjust the beam size at the sample and to reduce parasitic scattering. The diffraction patterns in the small- and wide-angle regime were recorded simultaneously using two sealed linear detectors with delay line readout connected in series [23]. The distance between the sample and the small-angle detector was 230 cm, and the sample to wide-angle detector distance was 50 cm. The reciprocal spacings s = (2/A)sin 0 (A wavelength of radiation, 20 scattering angle) were calibrated in the small-angle region by the diffraction pattern of collagen, whereas for the wide-angle region p-bromo-benzoic acid was used. Tlte range of scattering vectors covered is particularly well suited for the study of small and large scale structures of polymers up to a typical unit size of about 300 A. 1-Monoolein and ~-lactoglobulin were obtained from Sigma Chemical Co. (Deisenhofen, Germany), dioleoylphosphatidylethanolamine (DOPE) and dipalmitoylphosphatidylcholine (DPPC) were obtained from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). The substances had a purity of >99% and were used without further treatment. The lipid-water dispersions with a lipid concentration of about 20% (w/w) were subjected to three vortex-freeze-thaw cycles. The 10% (w/w) P-lactoglobulin solution was prepared by dissolving the dry protein in buffer solution (t~s[hydroxymethyl]~inometh~e, pH 7.4). To illustrate the potential of the experimental setup, we present examples of different lipid systems and of a watersoluble protein.
3. Results and discussion _
SAXS-detector
detector
monochromotor
17 r------23Ocm
beam
sample -
II n slits
DORIS
Fig. I. Schematic drawing of the experimentai setup for the of the synchrotron radiation, DAC with tbe opposed diamond anvil configuration and a metal foil gasket, and detector system at beamline X13 at the EMBL outstation at DESY in combination
Hamburg (SAXS small-angle X-ray scattering, WAXS wide-angle X-ray scattering). A blow-up of the sample environment is shown in the upper part (the different items shown are on vastly different scales).
3. I. Lipid dispersions Lipids constitute the basic structural component of biological membranes. Due to their amphiphilic nature most lipids form spontaneously lamellar structures when dispersed in water 1241. They are built up by stacks of layers consisting alternately of a lipid bilayer and a water layer. The polar headgroups of the lipid molecules are in contact with water whereas the apolar hydrocarbon chains are directed to the centre of the lipid bilayer. Most phospholipid bilayers exhibit several thermotropic lame&r phase transfo~ations. such as a gel to liquid-c~stalline phase transition. In the liquid-c~stalline phase, the hydrocarbon chains of the lipid bilayers are conformationally disordered, whereas in the gel phase the hydrocarbon chains are more extended and relatively ordered. Although most lipids in excess water exist in lamellar phases, certain lipids, such as phosphatidylethanol~~nes and mono-
849
C. C:eulL et nl. I Nucl. Instr. and Mtltll. in Phys. Rrs. A 368 (1996) 847-US1
acylglycerides, can form an inverted hexagonal (H,,) or cubic phases 125.261. The H,,-phase consists of infinite water cylinders su~ounded by lipid molecules, packed in a two-dimensional hexagonal lattice. The cubic phases consist of continuous regions of water and hydroc~bon, which can be described by infinite periodic minimal surfaces. These are intersection-free surfaces periodic in three dimensions with a mean curvature that is everywhere zero. Nonlamellar lipid phases possibly play an important functional role in some cell processes as local and transient intermediates [25,26]. The temperature and water concentratian dependent phase behaviour is well known for many lipids (see, e.g., Refs. [23-291). To explore the complete temperature-pressure phase diagram of aqueous lipid dispersions, the diamond anvil technique is an adequate tool. To illustrate the Performance of the experimental setup a diffraction pattern of a lipid-water dispersion (20 wt.% DQPE. 22°C) in the diamond anvil cell is shown in Fig. 2. The first three orders of reflection with Miller indices ( lo), ( 11) and (20) of the inverted hexagonal lipid phase are observed. The background scattering (DAC filled with water) is also shown for comparison. The latter is relatively low in the s-range under study and thus allows to obtain high quality diffraction patterns in this high pressure sample environment. Small- and wide-angle diffraction patterns of the monoacylglyceride I-monoolein (MO) at different pressures are shown in Fig. 3. At 1 bar six Bragg peaks are seen in the small-angle region which are positioned in the ratio -\/2:~3:t’4:~66:t:~9. They can be indexed as the i 110). (Ill), (200), (211). (220), and (221) reflections on a cubic primitive lattice of space group Pn3m. Its lattice constant a is related to the reciprocal spacing s by s = (I ‘(h’ + #+’+ I’)“‘, where (hkl) are the Miller indices. The lattice constant a of the cubic phase increases from
11000 bar
9300 bar 6300 bar 4700 bar --@‘-N-+++++-
0
0.01 0.02 0.03
’ ,a-
0.04 0.2 s CA’1
3000 bar 1 bar 0.3
Fig. 3. Diffraction patterns of a 20 wt.% MO-water dispersion in the small- and wide-angle region at 7’- 21°C at different preSS”reS.
106 to 110 A upon a pressure increase to 3 kbar. This is the consequence of the ordering effect pressure imposes on the conformation of the acyl chains, which results in a decrease of the curvature of the lipid monolayers and thus to an increase of the lattice constant n. In the wide-angle region, a broad diffraction maximum typical for a disordered liquid-like packing of the acyl chains is observed at about (4.5 A)- ‘. Increasing the pressure to 4.7 kbar induces a transition to a lamellar phase with smaller partial molar lipid volume. The first lamellar reflection (100) observed at 0.0236 A corresponds to a lamellar lattice constant of 42 A. The lamellar repeat unit consists of the thickness of the lipid bilayer plus the interlamellar water layer around the lipid headgroups. At 4.7 kbar and above multiple Bragg reflections occur in the wide-angle region indicating a crystalline-like packing of the lipid chains. As an example of a lamellar phospholipid system. the pressure dependent structure of a 20 wt.% DPPC-water dispersion was studied at 23°C. Fig. 4 shows first-order Bragg reflections of different lamellar gel phases of DPPC up to 17 kbar. The lamellar lattice constant, which is 63 A at I bar, decreases to about 61 A at p = 11 kbar. At higher pressures a drastic drop in lamellar repeat spacing to a = 55 A occurs. At these pressures, most of the water contained in the multilamellar lipid vesicle freezes IS]. Interestingly, the ice is not found between the bilayers. it probably exists as a pool of crystalline ice in equilibrium
(11)
_112_
background
(20)
o-‘h
t 0
*
1
*
1
0.01
0.02 s
CA”]
*
1
*
0.03
Fig. 2. Diffmction patterns of a 20 wt.% DOPE-water and the background of the DAC at 22°C.
0. dispersion
Fig. 4. Selected diffraction patterns of a 20 wt.% DPPC-wafer dispersion in the small- and wide-angle region at T = 23°C and different pressures.
850
C. Czeslik et al.
/ Nucl. Instr.
and Meth.
with the bound water still associated with the polar lipid headgroups. The multilamellar lipid assembly does not seem to be significantly destroyed by the freezing of the solvent. Information about the packing of the lipid molecules is obtained from the wide-angle diffraction pattern. At ambient pressure one peak isOobserved around (4.17 A))’ with a shoulder at (4.08 A)- ‘. This diffraction pattern corresponds to the packing of the lipid molecules in a distorted hexagonal lattice with the hydrocarbon chains tilted with respect to the lipid bilayer normal. At pressures above 3 kbar the shoulder in the wide-angle region has disappeared, indicating that the acyl chains are probably no longer tilted. The wide-angle peak shifts continuously from (4.09 A)-’ at 3 kbar to (3.76 A)-’ at 19 kbar. The lateral lipid packing increases signi~cantly even up to these extreme pressures. The calculated unit cell compressibility is about 1.3 X 10d5 bar-‘. 3.2. Protein sokions Pressure-induced denaturation of a protein was first observed by Bridgman in 1914. but systematic studies of the effect only began half a century later. Bridgman observed that egg white coagulates between 5 and 7 kbar [30]. It is believed that protein denaturation is mainly caused by rearrangement and/or destruction of non-covalent bonds, such as hydrogen and ionic bonds and hydrophobic interaction in the tertiary and quarternary structure of proteins. Pressure denaturation instead of heat denaturation has not been used in food processing until recently [3]. It has been shown that the texture of the pressure-induced gels may drastically differ from that of
in Phys. Res. A 368 (1996)
847-851
beat-induced gels suggesting that the underlying gelation mechanism may be different. We studied the globular protein @lactoglobulin, a major whey protein with a molecular mass of about 36 kDa and * radius of gyration Rs of about ‘21 A [31,32]. In the native state at room temperature and pH = 7, the protein solution structure mainly consists of dimers and tetramers. Upon denaturation at high temperature (above about 70°C) the solution structure is unstable and aggregation occurs [31]. The development of the network results in gelation, such as is observed with heat-denatured egg white. Fig. 5 exhibits small-angle scattering curves of a 10 wt.% P-lactoglobulin solution at 1 bar and 9 kbar and T = 23°C. For comparison, the scattering curve of the pure buffer in the DAC is also shown. The radius of gyration of the protein at ambient pressure, which has been determined from the Guinier-plot. is about 29 A, indicating that the protein structure consists mainly of tetrameric units. At 9 kbar the scattering intensity in the small-angle region has changed drastically due to the pressure-induced denaturation and gel formation of the protein. The analysis (slope of the In i(s) vs. In s plot) of the intermediate s-range reveals that the protein gel exhibits volume fractal-like structural properties with a fractal dimension of D = 2.7. The examples presented above serve to demons~ate that the experimental setup is suitable for perfoming simultaneously low- and wide-angle X-ray diffraction studies of biomolecules in the DAC, so that valuable information can be obtained about their structure and phase transformations at elevated pressures. Furthermore. this technique also allows to study the effect of ice formation on biomolecular structure at superzero temperatures and high pressures. This subject may be of considerable biological and tech-
I
9-
80 9000bar + lbar 0 background
765432l0
0.006
0.01 s CA-~]
Fig. 5. Small-angle scattering curves of a 10 wt.% ~-lactoglobul~n the background scattering curve is also given.
0.014
0.018
solution in the DAC at T= 23°C and selected pressures.
For comparison,
nological importance, especially for studying osmotic dehydration, cold-induced denaturation and cryoprotection phenomena..
Acknowledgments We thank Dr. Jens Otto for providing access to his diode array spectrometer for pressure calibration and Dr. M.H.J. Koch for helpful discussions. We are grateful to the DFG for financial support.
References K. Heremans. Ann. Rev. Biophys. Bioeng. I I (1982) 1. P.T.T. Wong, in: High Pressure Chemistry and Biochemistry, eds. R. van Eldik, J. Jonas (D. Reidel, 1987) p. 381. C. Balny, R. Hayashi, K. Heremans and P. Masson (eds.), High Pressure and Biotechnology (John Libbey Eurotext, 1992). R. Winter and J. Jonas (eds.), High Pressure Chemistry, Biochemistry and Materials Science (Kluwer, Dordrecht, The Netherlands. 1993). VV. Mozhaev, K. Heremans, 1. Frank. P. Masson and C. Balny, TJBTECH 12 (1994) 493. M. Gross and R. Jaenicke. Europ. J. Biochem. 221 (1994) 617. R. Winter, A. Landwehr, T. Brauns, J. Erbes, C. Czeslik and 0. Reis, in: Proc. 23rd Steenbock Symp. on High Pressure Effects in Molecular Biophysics and Enzymology (Madison, USA, 1994). C.W.F.T. Pistorius, E. Rapoport and J. B. Clark, J. Chem. Phys. 48 ( 1968) 5509. l?L.-G. Chong and G. Weber, Biochemisty 22 ( 1983) 5544. SK. Prasad, R. Shashidhar, B.P. Gaber and SC. Chandrasekhar, Chem. Phys, Lip. 143 (1987) 227. D. Driscoll, J. Jonas and A. Jonas, Chem. Phys. Lip. 58 (1991) 97.
iI21 X. Peng, A. Jonas and J. Jonas, Biophys. J. 68 (1995) 1137. (131 S. Utoh and T. Takemura, Jpn. 3. Appl. Phys. 24 (1985) 1404. Biochemisty 25 (1986) [I41 L.F. Braganza and D.L.Worcester, 2591. 1151 R. Winter and W.-C. Pilgrim. Ber. Bunsenges. Phys. Chem. 93 ( 1989) 708. iI61 R. Winter and P. Thiyagarajan, Progr. Polym. Sei. 8 1 C1990t 216. [I71 P.T.C. So, S.M. Gruner and E. Shyamsunder. Rev. Sci. lnstr. 63 (1992) 1763. I181 A. Landwehr and R. Winter. Ber. Bunsenges. Phys. Chem. 98 (1994) 214. I191 P.T.T. Wong, D.J. Siminovitch and H.H. Mantsch. Biochim. Biophys. Acta 947 ( 1988) 139. WI A. Jayaraman, Rev. Mod. Phys. 55 ( 1983) 65. Expe~mental TechPi1 W,F. Sherman and A.A. S~dtmulier. niques in High Pressure Research (Wiley, New York, 1987). p-21 D.J. Dunstan and I.L. Spain. 3. Phys. E 22 f 1989) 913: ibid 1923. u31 G. Rapp, A. Gabriel, M. Dositre and M.H.J. Koch, Nucl. Instr. and Meth., in press. ~41 G. Cevc and D. Marsh. Phospholipid Bilayers (Wiley, New York. 1987). 1251 J.M. Seddon, Biochim. Biopbys. Acta 1031 ( 1990) i. Biophys. Acta 988 WI G. Lindblom and L. Rilfors. B&him. (1989) 221. ~71 J. Briggs and M. Caffrey, Biophys. J. 66 (1994) S73. WI C. Czesiik, R. Winter, G. Rapp and K. Bartels, Biophys. J.. 68 (1995) 1423. ~91 J. Erbes. C. Czeslik, W. Hahn, R. Winter, M. Rappolt and G. Rapp. Ber. Bunsenges. Phys. Chem. 98 ( 1994) 1287. [30] P.W. Bridgman. J. Biol. Chem. 19 (1914) 511. [31j J. Witz. S.N. Timasheff and V. Luzzati, J. Am. Chem. Sot. 86 (1964) 168. [32] W. G. Griffin, M. C. A. Griffin. S. R. Martin and J. Price. J. Chem. Sot. Faraday Trans. 89 t 1993) 3395.
NUCLeUI INSTMMENTS &METnooS IN PHYSICS “Y!Z!?”
EISEVIER
High pressure synchrotron X-ray diffraction studies of biological molecules using the diamond anvil technique C. Czeslik”, R. Malessa”, R. Winter”.*, G. Rapph
Received
13
April 1995; revised
form received 2 August 1995
Abstract A system for high pressure synchrotron X-ray diffraction studies of biological samples in a diamond anvil cell (DAC) is described. It is capable of operating in the whole temperature and pressure range of interest for studies of biological molecules, i.e.. in the temperature range from -40 to IOO”C at pressures between 1 bar and 50 kbar. The pressure is calibrated by measuring the pressure dependence of the ruby fluorescence line at 694 nm. Two linear detectors connected in series are used to measure simultaneously the small- and wide-angle X-ray scattering. The advantage of the experimental technique is threefold: Firstly, the amount of sample can be kept to a minimum (ea. 30 nl) using the high intensity of synchrotron radiation. Secondly, only the diamond anvil technique allows to reach extreme pressures. Thirdly, the use of the dual detector system allows recording of diffraction data both in the small- and wide-angle region at the same time. Examples of hitherto unknown phases of aqueous lipid and protein samples illustrate the potential of the system.
1. Intr~uction In recent years considerable effort has been put into studying pressure effects on bio~hemicai systems [l-7]. Besides the physico-chemical interest in using pressure as a thermodynamic variable to understand the structure. phase behaviour and dynamics of biological molecules and to develop and prove theoretical approaches, high pressure is also of considerable physiological and biotechnological interest. For example. pressure studies are of interest to understand the physiology of deep sea organisms (in the deep sea pressures up to 1.2 kbar occur), the sensitivity of excitable cell membranes to pressure (“high pressure nervous syndrom”), and the antagonistic effect of pressure to anesthetic action. In food technology. application of high hydrostatic pressure in food sterilization and processing has been a central issue of research and development in Japan since several years [3]. Due to pressure-induced ice formation. the pressure range for studying biomolecules is mainly restricted to pressures below 20 kbar IS]. Up to this pressure, the main effects of applied pressure derive from changes of intermolecular distances, which generally lead to conformational changes, but also from ionization. aggregation and dissociation of biological molecules. The structure and * Corresponding
author. ‘Tel. 1-49 23 1 755 3900. fax +49
7-3 I
755 3901,
016%9001/9&/$15.00 SSD/
0
0
16%9003( 95
1996 Elsevier Science B.V. All rights reserved )OO78
l-1
stability of biological molecules depend mainly on three kinds of interactions: ionic. hydrophobic and hydrogen bonding. Due to the electrostrictive effect of separated charges, ion pairs in aqueous solution are strongly stabilized by hydrostatic pressure. The pH of water decreases with pressure and the exposure of hydrophobic residues, occuring for instance during unfolding of proteins, is favoured by elevated pressures. Hydrogen bond formation in biomolecules is generally accompanied by a negligibly small positive or negative volume change. Considering the fundamental principles of the solution structure of globular proteins. i.e., optimum packing of the hydrophobic core. minimum hydrophobic surface area, and formation of ion pairs within and between subunits. it is evident that hydrostatjc pressure below 20 kbar mainly affects both the tertiary and quatemary structure of proteins. In the past thermodynamic and spectroscopic methods have been widely used for studies on high pressure phase behaviour and structure of biomolecules. and these studies were restricted to pressures below 3-S kbar (see. e.g.. Refs. 19-181). Only recently. the high-pressure FI’IR technique has been developed for measurements at higher pressures 12,191. X-ray and neutron diffraction work was also limited to about 4 kbar in the past [ 14- 171. The availability of high flux synchrotron X-ray sources and recent advances in X-ray detector development, in conjunction with the diamond anvil technique. allow to extend the pressure range up to extreme pressures. Although the
848
C. Czeslik et al. I Nucl. Instr. and Meth. in Phys. Res. A 368 (1996) 847-851
DAC is already a widely used tool for diffraction work in research of the solid state [20], its potential for studies of biological solution structures at elevated pressures has not been recognized so far. We present a setup for studying simultaneously the small- and wide-angle diffraction pattern of biomolecules and biomolecul~ aggregates at pressures up to 50 kbar, and its performance is illustrated by examples of recent experiments.
2. Experimental setup A diamond anvil cell with type IIa diamonds (Diamond Optics, Tucson, USA) was used for the high pressure X-ray diffraction experiments. The sample under study is positioned between the two diamonds (see Fig. 1) which have a cut with 16 facets. The diameter of the anvil surface is 0.6 mm. The total path length of the X-ray beam through the diamonds is about 5 mm, which leads to a reduction of the primary photon intensity by a factor of about lo4 (at A = 1.5 A). The sample is sealed in a gasket (made of Inconel 718 with 53% Ni. 19% Fe, and 19% Cr) of 0.25 mm thickness with a hole of 0.37 mm diameter. The total volume of the sample is about 30 nl. The diamond anvils are held in a thermostated jacket. Since the sample volume is very small and the absorption of the X-ray intensity by the diamonds is very high, the diffracted intensity is extremely weak and exposure times of several minutes (typically 10 min) are required at common synchro~on X-ray sources. Pressure is calibrated by measuring the shift of the fluorescence line at 694 nm of a small ruby chip which is added to the sample in the gasket hole (dhldp = 0.036 nm/kbar [21,22]).
The small- and wide-angle X-ray diffraction experiments were performed at beamline X13 of the EMBL outstation at DESY in Hamburg. With the germanium monochromator installed, the wavelength of the X-rays is fixed to 1.5 A. Sets of tungsten slits are used to adjust the beam size at the sample and to reduce parasitic scattering. The diffraction patterns in the small- and wide-angle regime were recorded simultaneously using two sealed linear detectors with delay line readout connected in series [23]. The distance between the sample and the small-angle detector was 230 cm, and the sample to wide-angle detector distance was 50 cm. The reciprocal spacings s = (2/A)sin 0 (A wavelength of radiation, 20 scattering angle) were calibrated in the small-angle region by the diffraction pattern of collagen, whereas for the wide-angle region p-bromo-benzoic acid was used. Tlte range of scattering vectors covered is particularly well suited for the study of small and large scale structures of polymers up to a typical unit size of about 300 A. 1-Monoolein and ~-lactoglobulin were obtained from Sigma Chemical Co. (Deisenhofen, Germany), dioleoylphosphatidylethanolamine (DOPE) and dipalmitoylphosphatidylcholine (DPPC) were obtained from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). The substances had a purity of >99% and were used without further treatment. The lipid-water dispersions with a lipid concentration of about 20% (w/w) were subjected to three vortex-freeze-thaw cycles. The 10% (w/w) P-lactoglobulin solution was prepared by dissolving the dry protein in buffer solution (t~s[hydroxymethyl]~inometh~e, pH 7.4). To illustrate the potential of the experimental setup, we present examples of different lipid systems and of a watersoluble protein.
3. Results and discussion _
SAXS-detector
detector
monochromotor
17 r------23Ocm
beam
sample -
II n slits
DORIS
Fig. I. Schematic drawing of the experimentai setup for the of the synchrotron radiation, DAC with tbe opposed diamond anvil configuration and a metal foil gasket, and detector system at beamline X13 at the EMBL outstation at DESY in combination
Hamburg (SAXS small-angle X-ray scattering, WAXS wide-angle X-ray scattering). A blow-up of the sample environment is shown in the upper part (the different items shown are on vastly different scales).
3. I. Lipid dispersions Lipids constitute the basic structural component of biological membranes. Due to their amphiphilic nature most lipids form spontaneously lamellar structures when dispersed in water 1241. They are built up by stacks of layers consisting alternately of a lipid bilayer and a water layer. The polar headgroups of the lipid molecules are in contact with water whereas the apolar hydrocarbon chains are directed to the centre of the lipid bilayer. Most phospholipid bilayers exhibit several thermotropic lame&r phase transfo~ations. such as a gel to liquid-c~stalline phase transition. In the liquid-c~stalline phase, the hydrocarbon chains of the lipid bilayers are conformationally disordered, whereas in the gel phase the hydrocarbon chains are more extended and relatively ordered. Although most lipids in excess water exist in lamellar phases, certain lipids, such as phosphatidylethanol~~nes and mono-
849
C. C:eulL et nl. I Nucl. Instr. and Mtltll. in Phys. Rrs. A 368 (1996) 847-US1
acylglycerides, can form an inverted hexagonal (H,,) or cubic phases 125.261. The H,,-phase consists of infinite water cylinders su~ounded by lipid molecules, packed in a two-dimensional hexagonal lattice. The cubic phases consist of continuous regions of water and hydroc~bon, which can be described by infinite periodic minimal surfaces. These are intersection-free surfaces periodic in three dimensions with a mean curvature that is everywhere zero. Nonlamellar lipid phases possibly play an important functional role in some cell processes as local and transient intermediates [25,26]. The temperature and water concentratian dependent phase behaviour is well known for many lipids (see, e.g., Refs. [23-291). To explore the complete temperature-pressure phase diagram of aqueous lipid dispersions, the diamond anvil technique is an adequate tool. To illustrate the Performance of the experimental setup a diffraction pattern of a lipid-water dispersion (20 wt.% DQPE. 22°C) in the diamond anvil cell is shown in Fig. 2. The first three orders of reflection with Miller indices ( lo), ( 11) and (20) of the inverted hexagonal lipid phase are observed. The background scattering (DAC filled with water) is also shown for comparison. The latter is relatively low in the s-range under study and thus allows to obtain high quality diffraction patterns in this high pressure sample environment. Small- and wide-angle diffraction patterns of the monoacylglyceride I-monoolein (MO) at different pressures are shown in Fig. 3. At 1 bar six Bragg peaks are seen in the small-angle region which are positioned in the ratio -\/2:~3:t’4:~66:t:~9. They can be indexed as the i 110). (Ill), (200), (211). (220), and (221) reflections on a cubic primitive lattice of space group Pn3m. Its lattice constant a is related to the reciprocal spacing s by s = (I ‘(h’ + #+’+ I’)“‘, where (hkl) are the Miller indices. The lattice constant a of the cubic phase increases from
11000 bar
9300 bar 6300 bar 4700 bar --@‘-N-+++++-
0
0.01 0.02 0.03
’ ,a-
0.04 0.2 s CA’1
3000 bar 1 bar 0.3
Fig. 3. Diffraction patterns of a 20 wt.% MO-water dispersion in the small- and wide-angle region at 7’- 21°C at different preSS”reS.
106 to 110 A upon a pressure increase to 3 kbar. This is the consequence of the ordering effect pressure imposes on the conformation of the acyl chains, which results in a decrease of the curvature of the lipid monolayers and thus to an increase of the lattice constant n. In the wide-angle region, a broad diffraction maximum typical for a disordered liquid-like packing of the acyl chains is observed at about (4.5 A)- ‘. Increasing the pressure to 4.7 kbar induces a transition to a lamellar phase with smaller partial molar lipid volume. The first lamellar reflection (100) observed at 0.0236 A corresponds to a lamellar lattice constant of 42 A. The lamellar repeat unit consists of the thickness of the lipid bilayer plus the interlamellar water layer around the lipid headgroups. At 4.7 kbar and above multiple Bragg reflections occur in the wide-angle region indicating a crystalline-like packing of the lipid chains. As an example of a lamellar phospholipid system. the pressure dependent structure of a 20 wt.% DPPC-water dispersion was studied at 23°C. Fig. 4 shows first-order Bragg reflections of different lamellar gel phases of DPPC up to 17 kbar. The lamellar lattice constant, which is 63 A at I bar, decreases to about 61 A at p = 11 kbar. At higher pressures a drastic drop in lamellar repeat spacing to a = 55 A occurs. At these pressures, most of the water contained in the multilamellar lipid vesicle freezes IS]. Interestingly, the ice is not found between the bilayers. it probably exists as a pool of crystalline ice in equilibrium
(11)
_112_
background
(20)
o-‘h
t 0
*
1
*
1
0.01
0.02 s
CA”]
*
1
*
0.03
Fig. 2. Diffmction patterns of a 20 wt.% DOPE-water and the background of the DAC at 22°C.
0. dispersion
Fig. 4. Selected diffraction patterns of a 20 wt.% DPPC-wafer dispersion in the small- and wide-angle region at T = 23°C and different pressures.
850
C. Czeslik et al.
/ Nucl. Instr.
and Meth.
with the bound water still associated with the polar lipid headgroups. The multilamellar lipid assembly does not seem to be significantly destroyed by the freezing of the solvent. Information about the packing of the lipid molecules is obtained from the wide-angle diffraction pattern. At ambient pressure one peak isOobserved around (4.17 A))’ with a shoulder at (4.08 A)- ‘. This diffraction pattern corresponds to the packing of the lipid molecules in a distorted hexagonal lattice with the hydrocarbon chains tilted with respect to the lipid bilayer normal. At pressures above 3 kbar the shoulder in the wide-angle region has disappeared, indicating that the acyl chains are probably no longer tilted. The wide-angle peak shifts continuously from (4.09 A)-’ at 3 kbar to (3.76 A)-’ at 19 kbar. The lateral lipid packing increases signi~cantly even up to these extreme pressures. The calculated unit cell compressibility is about 1.3 X 10d5 bar-‘. 3.2. Protein sokions Pressure-induced denaturation of a protein was first observed by Bridgman in 1914. but systematic studies of the effect only began half a century later. Bridgman observed that egg white coagulates between 5 and 7 kbar [30]. It is believed that protein denaturation is mainly caused by rearrangement and/or destruction of non-covalent bonds, such as hydrogen and ionic bonds and hydrophobic interaction in the tertiary and quarternary structure of proteins. Pressure denaturation instead of heat denaturation has not been used in food processing until recently [3]. It has been shown that the texture of the pressure-induced gels may drastically differ from that of
in Phys. Res. A 368 (1996)
847-851
beat-induced gels suggesting that the underlying gelation mechanism may be different. We studied the globular protein @lactoglobulin, a major whey protein with a molecular mass of about 36 kDa and * radius of gyration Rs of about ‘21 A [31,32]. In the native state at room temperature and pH = 7, the protein solution structure mainly consists of dimers and tetramers. Upon denaturation at high temperature (above about 70°C) the solution structure is unstable and aggregation occurs [31]. The development of the network results in gelation, such as is observed with heat-denatured egg white. Fig. 5 exhibits small-angle scattering curves of a 10 wt.% P-lactoglobulin solution at 1 bar and 9 kbar and T = 23°C. For comparison, the scattering curve of the pure buffer in the DAC is also shown. The radius of gyration of the protein at ambient pressure, which has been determined from the Guinier-plot. is about 29 A, indicating that the protein structure consists mainly of tetrameric units. At 9 kbar the scattering intensity in the small-angle region has changed drastically due to the pressure-induced denaturation and gel formation of the protein. The analysis (slope of the In i(s) vs. In s plot) of the intermediate s-range reveals that the protein gel exhibits volume fractal-like structural properties with a fractal dimension of D = 2.7. The examples presented above serve to demons~ate that the experimental setup is suitable for perfoming simultaneously low- and wide-angle X-ray diffraction studies of biomolecules in the DAC, so that valuable information can be obtained about their structure and phase transformations at elevated pressures. Furthermore. this technique also allows to study the effect of ice formation on biomolecular structure at superzero temperatures and high pressures. This subject may be of considerable biological and tech-
I
9-
80 9000bar + lbar 0 background
765432l0
0.006
0.01 s CA-~]
Fig. 5. Small-angle scattering curves of a 10 wt.% ~-lactoglobul~n the background scattering curve is also given.
0.014
0.018
solution in the DAC at T= 23°C and selected pressures.
For comparison,
nological importance, especially for studying osmotic dehydration, cold-induced denaturation and cryoprotection phenomena..
Acknowledgments We thank Dr. Jens Otto for providing access to his diode array spectrometer for pressure calibration and Dr. M.H.J. Koch for helpful discussions. We are grateful to the DFG for financial support.
References K. Heremans. Ann. Rev. Biophys. Bioeng. I I (1982) 1. P.T.T. Wong, in: High Pressure Chemistry and Biochemistry, eds. R. van Eldik, J. Jonas (D. Reidel, 1987) p. 381. C. Balny, R. Hayashi, K. Heremans and P. Masson (eds.), High Pressure and Biotechnology (John Libbey Eurotext, 1992). R. Winter and J. Jonas (eds.), High Pressure Chemistry, Biochemistry and Materials Science (Kluwer, Dordrecht, The Netherlands. 1993). VV. Mozhaev, K. Heremans, 1. Frank. P. Masson and C. Balny, TJBTECH 12 (1994) 493. M. Gross and R. Jaenicke. Europ. J. Biochem. 221 (1994) 617. R. Winter, A. Landwehr, T. Brauns, J. Erbes, C. Czeslik and 0. Reis, in: Proc. 23rd Steenbock Symp. on High Pressure Effects in Molecular Biophysics and Enzymology (Madison, USA, 1994). C.W.F.T. Pistorius, E. Rapoport and J. B. Clark, J. Chem. Phys. 48 ( 1968) 5509. l?L.-G. Chong and G. Weber, Biochemisty 22 ( 1983) 5544. SK. Prasad, R. Shashidhar, B.P. Gaber and SC. Chandrasekhar, Chem. Phys, Lip. 143 (1987) 227. D. Driscoll, J. Jonas and A. Jonas, Chem. Phys. Lip. 58 (1991) 97.
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