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Dynamics of ganglioside micellar solutions by quasielastic neutron scattering
ARTICLE IN PRESS
Physica B 350 (2004) e619–e622
Dynamics of ganglioside micellar solutions by quasielastic neutron scattering P. Broccaa, L. Cantu" a, F. Cavatortab, M. Cortia, E. Del Faveroa, A. Deriub, M. Di Barib,* a
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita" di Milano and Istituto Nazionale per la Fisica della Materia, L.I.T.A., via F.lli Cervi 93, I-20090 Segrate, Milano, Italy b Dipartimento di Fisica, Universita" di Parma and Istituto Nazionale per la Fisica della Materia, Parco Area delle Scienze 7/A, I-43100 Parma, Italy
Abstract Gangliosides are double-tailed biological amphiphiles naturally abundant in the nervous system. We present a quasielastic neutron scattering study on the dynamics of ganglioside molecules in 15% concentration micellar solution. The scattering contribution due to gangliosides has been analysed in terms of a simple model of confined diffusion within a sphere with rigid walls. r 2004 Elsevier B.V. All rights reserved. PACS: 61.12.q; 82.70.Uv; 82.30.Rs Keywords: Gangliosides; Ganglioside dynamics; Neutron scattering
Gangliosides are double-tailed biological amphiphiles with a complex polar headgroup formed by several sugar units. They are naturally abundant in the nervous system, where they are known to play a role in recognition processes and in the transmembrane information transfer [1]. When dissolved in water above a very low critical concentration (E108 M), they form micelles having the shape of an oblate ellipsoid of revolution with average hydrodynamic radius ( and aggregation number N of the RH=50–60 A order of few hundreds [2]. The most typical *Corresponding author. Tel.: +39-0521-905244 ext. 561; fax: +39-0521-905223. E-mail addresses: dibari@fis.unipr.it, mariateresa.dibari@fis.unipr.it (M. Di Bari).
representative of this class of molecules is the GM1 ganglioside. It bears five sugar units in its headgroup, one of which is a sialic acid residue. In solution, GM1 micelles are in a disordered L1 phase up to 30% by weight [3]. Although being charged molecules, their packing, in the micellar aggregate, is dictated by steric requirements, due to the huge geometric hindrance of the headgroup. GM1 micelles present a bistable behaviour between stable states (hereafter called A and B); the transition between the two states is triggered by some external agent, like temperature [4,5]. At low temperatures, GM1 micelles can be found with an aggregation number of either 300 (state A) or 220 (state B), according to their thermal history. The fact that GM1 micelles can exist with two stable average dimensions, is a signature of two different
0921-4526/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2004.03.165
ARTICLE IN PRESS e620
P. Brocca et al. / Physica B 350 (2004) e619–e622
packing geometries which can be assumed by the individual GM1 molecule within the aggregate, with a larger area per headgroup in state B than in state A. Then, the ganglioside molecular hindrance is modified by the conformational transition, for example the oligosaccharide chain axis may tilt with respect to the ceramide portion [6]. This rearrangement could involve a different level of hydration, by changing the relative interplay of the hydrogen bonding of the NH group in the longchain base and the glycosidic oxygen within the same molecule. We have initiated a systematic study of the dynamics of water in ganglioside micellar solutions. First neutron experiments carried out by us indicated that the presence of ganglioside micelles slows down the dynamics of the solvent with respect to that of pure water and that its mobility is lightly higher in the A state than in the B state [7]. The present study addresses the dynamics of ganglioside molecules themselves. To this purpose GM1 micelles were prepared in the B state and measurements were carried out in a deuterated buffer in order to minimise the contribution from water molecules. Solutions were prepared by dissolving GM1 in pure D2O to a final volume fraction of 15%. They were then put into flat quartz cells 50 30 mm, 0.5 mm thick. QENS measurements were carried out on the backscattering spectrometer IRIS at the ISIS neutron pulsed source. IRIS was operated in the PG002 analyser configuration providing an energy resolution DE=14 meV (FWHM) in the ( 1. The spectra were analysed Q-range 0.44–1.84 A in the energy range 0.3 to +1.5 meV in terms of the following dynamical structure factor:
the micelle as a whole [8] Sgan ðQ; oÞ ¼ ½A0 ðQÞdðoÞ þ ð1 A0 ðQÞÞLgan ðQ; oÞ
#LCM ðQ; oÞ: ð2Þ A0(Q) is the elastic incoherent structure factor, EISF, and Lgan(Q,o) is a quasielastic component described by a single Lorentzian term. For the scattering law of the buffer, we adopted a simple phenomenological model already used to describe QENS data from pure supercooled water [9] Sbuf ðQ; oÞ ¼ ½A1 ðQÞL1 ðQ; oÞ þ ð1 A1 ðQÞÞL2 ðQ; oÞ :
ð3Þ
It contains two Lorentzian components: a narrow one, L1, with weight A1(Q), that describes the slow relaxation processes corresponding to the longrange translational diffusion, and a broad one, L2, that accounts for the local dynamics of water molecules. The total resulting scattering law has then been convoluted to the instrumental resolution function. In the fit the diffusion coefficient of the micelles that determines the width of LCM(Q,o) is kept fixed to the value D0=1.8 107 cm2 s1 deduced from the hydrodynamic radius of the micelles. A typical quasielastic spectrum obtained for a 15% ganglioside solution in D2O buffer is shown in Fig. 1. It can be seen that the quasielastic component due to ganglioside hydrogens is much
2 2 Stot ðQ; oÞ ¼ eQ hu i=3 ½Agan Sgan ðQ; oÞ
þ ð1 Agan ÞSbuf ðQ; oÞ þ B:
ð1Þ
A common Debye–Waller factor, given by exp(Q2/u2S)/3 was assumed to take into account fast vibrational motions. Agan is the scattering intensity from gangliosides and from the associated water, and B is a flat instrumental background. The ganglioside contribution is assumed to be described by a confined diffusive motion; it is then convoluted to a Lorentzian LCM(Q,o) that describes the Brownian diffusion of
( 1 for a 15% GM1 Fig. 1. QENS spectrum at Q=1.3 A ganglioside micellar solution in D2O. The total fit as well as the components due to the ganglioside (Iel and IQENS) and to the buffer (continuous line: slow relaxation, and dashed line: fast relaxation) are reported.
ARTICLE IN PRESS P. Brocca et al. / Physica B 350 (2004) e619–e622
narrower than that of the buffer and it can be determined with good accuracy with the adopted instrumental configuration, while the buffer contribution, in the same energy window, is close to a flat background. From a first data fit with free parameters we find Agan=0.4870.1. This value is larger than the one (0.43) expected on the basis of the sample isotopic composition. We attribute this difference to the contribution of water molecules that are closely associated to the micelles, so that their dynamics cannot be distinguished from that of GM1 hydrogens. Agan was then kept fixed to 0.48 in all the subsequent fits. From the Q-dependence of the total scattering intensity we have derived a Debye–Waller-like factor and hence a value of the mean square ( 2 in atomic fluctuation: /u2S/3=(0.0870.02) A reasonable agreement with that measured for other biomolecules [10]. The diffusivity properties of the buffer, not surprisingly, are not identical to those of pure water, and correspond to a slightly reduced mobility. For the confined dynamics of ganglioside hydrogens we have adopted the simple Volino– Dianoux model of a free diffusion within a sphere with rigid walls [11]. The EISF can then be written as 3j1 ðQa Þ 2 Ao ðQÞ ¼ f þ ð1 f Þ ; ð4Þ Qa where j1(Qa) is the first-order spherical Bessel function, a is the radius of the confining sphere and f is the fraction of hydrogen atoms that do not take part to this confined diffusion. The Q-dependence of the EISF and its fit to Eq. (4) is shown in Fig. 2. From the fit we obtain a fraction f of immobile protons f=0.36, and a ( confining radius a=(2.470.2) A. The dynamics of the ganglioside molecule turns out to be quite slow. This is not unexpected, and it can be explained as a consequence of the rather close packing of the bulky sugar headgroups in the hydrophilic shell of the micelle. Also the estimated volume explored by protons is quite shallow ( from the EISF). Indeed it is lower than (R=2.4 A the one that could in principle be accessible, being the area per headgroup at the micelle interface
e621
Fig. 2. Elastic incoherent structure factor derived for the ganglioside contribution. The continuous curve is a fit to a Volino–Dianoux model of confined diffusion inside a rigid sphere.
( 2. One possibility could be that the overall B100 A average dynamics is made up of different contributions, coming from different portions of the molecule. In fact, when dealing with micelles, protons belonging to the hydrophobic core are usually considered as non-diffusing, thus contributing to the elastic part alone of the scattered intensity. This seems not be the case in GM1 micelles. Indeed the value of the fraction f of ‘immobile’ protons from the fit (f=0.36) is too small to account for all the hydrogens in the hydrophobic tails; under this hypothesis one would expect f=0.6. The arrangement of long double tails forced in a non-flat geometry by the hindrance of the headgroups could lead to significant protrusion effects, aided by the fact that the upper region of the chains, close to the headgroup, still contains groups with hydrophilic character. A portion of the hydrophobic moiety could then contribute to the quasielastic part of the scattered radiation, besides the saccharidic one. The resulting measured average dynamics would be lower than that of the headgroups alone. Another possibility is that the dynamics of the headgroup is effectively very slow and more confined than expected. This would lead us to conclude that a remarkably strong coordination occurs among solute and
ARTICLE IN PRESS e622
P. Brocca et al. / Physica B 350 (2004) e619–e622
solvent molecules. Further experiments are underway to clarify this point.
References [1] G. Tettamanti, et al., in: M. Corti, V. Degiorgio (Eds.), Physics of Amphiphiles: Micelles, Vesicles and Microemulsions, North-Holland, Amsterdam, 1985, p. 607. [2] S. Sonnino, et al., Chem. Phys. Lipids 71 (1994) 21.
" M. Corti, E. Del Favero, Physica A [3] M. Boretta, L. Cantu, 236 (1997) 162. " et al., Chem. Phys. Lipids 79 (1996) 137. [4] L. Cantu, " et al., J. Phys. (France) 6 (1996). [5] L. Cantu, [6] P.G. Nyholm, I. Pascher, Biochemistry 32 (1993) 1225. " et al., Physica B 234–236 (1997) 281. [7] L. Cantu, [8] J. Perez, J.-M. Zanotti, D. Durand, Biophys. J. 77 (1999) 454. [9] D. Di Cola, A. Deriu, M. Sampoli, A. Torcini, J. Chem. Phys. 104 (1996) 4223. [10] C. Andreani, et al., Biophys. J. 68 (1995) 2519. [11] F. Volino, A.J. Dianoux, Mol. Phys. 41 (1980) 271.
Physica B 350 (2004) e619–e622
Dynamics of ganglioside micellar solutions by quasielastic neutron scattering P. Broccaa, L. Cantu" a, F. Cavatortab, M. Cortia, E. Del Faveroa, A. Deriub, M. Di Barib,* a
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita" di Milano and Istituto Nazionale per la Fisica della Materia, L.I.T.A., via F.lli Cervi 93, I-20090 Segrate, Milano, Italy b Dipartimento di Fisica, Universita" di Parma and Istituto Nazionale per la Fisica della Materia, Parco Area delle Scienze 7/A, I-43100 Parma, Italy
Abstract Gangliosides are double-tailed biological amphiphiles naturally abundant in the nervous system. We present a quasielastic neutron scattering study on the dynamics of ganglioside molecules in 15% concentration micellar solution. The scattering contribution due to gangliosides has been analysed in terms of a simple model of confined diffusion within a sphere with rigid walls. r 2004 Elsevier B.V. All rights reserved. PACS: 61.12.q; 82.70.Uv; 82.30.Rs Keywords: Gangliosides; Ganglioside dynamics; Neutron scattering
Gangliosides are double-tailed biological amphiphiles with a complex polar headgroup formed by several sugar units. They are naturally abundant in the nervous system, where they are known to play a role in recognition processes and in the transmembrane information transfer [1]. When dissolved in water above a very low critical concentration (E108 M), they form micelles having the shape of an oblate ellipsoid of revolution with average hydrodynamic radius ( and aggregation number N of the RH=50–60 A order of few hundreds [2]. The most typical *Corresponding author. Tel.: +39-0521-905244 ext. 561; fax: +39-0521-905223. E-mail addresses: dibari@fis.unipr.it, mariateresa.dibari@fis.unipr.it (M. Di Bari).
representative of this class of molecules is the GM1 ganglioside. It bears five sugar units in its headgroup, one of which is a sialic acid residue. In solution, GM1 micelles are in a disordered L1 phase up to 30% by weight [3]. Although being charged molecules, their packing, in the micellar aggregate, is dictated by steric requirements, due to the huge geometric hindrance of the headgroup. GM1 micelles present a bistable behaviour between stable states (hereafter called A and B); the transition between the two states is triggered by some external agent, like temperature [4,5]. At low temperatures, GM1 micelles can be found with an aggregation number of either 300 (state A) or 220 (state B), according to their thermal history. The fact that GM1 micelles can exist with two stable average dimensions, is a signature of two different
0921-4526/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2004.03.165
ARTICLE IN PRESS e620
P. Brocca et al. / Physica B 350 (2004) e619–e622
packing geometries which can be assumed by the individual GM1 molecule within the aggregate, with a larger area per headgroup in state B than in state A. Then, the ganglioside molecular hindrance is modified by the conformational transition, for example the oligosaccharide chain axis may tilt with respect to the ceramide portion [6]. This rearrangement could involve a different level of hydration, by changing the relative interplay of the hydrogen bonding of the NH group in the longchain base and the glycosidic oxygen within the same molecule. We have initiated a systematic study of the dynamics of water in ganglioside micellar solutions. First neutron experiments carried out by us indicated that the presence of ganglioside micelles slows down the dynamics of the solvent with respect to that of pure water and that its mobility is lightly higher in the A state than in the B state [7]. The present study addresses the dynamics of ganglioside molecules themselves. To this purpose GM1 micelles were prepared in the B state and measurements were carried out in a deuterated buffer in order to minimise the contribution from water molecules. Solutions were prepared by dissolving GM1 in pure D2O to a final volume fraction of 15%. They were then put into flat quartz cells 50 30 mm, 0.5 mm thick. QENS measurements were carried out on the backscattering spectrometer IRIS at the ISIS neutron pulsed source. IRIS was operated in the PG002 analyser configuration providing an energy resolution DE=14 meV (FWHM) in the ( 1. The spectra were analysed Q-range 0.44–1.84 A in the energy range 0.3 to +1.5 meV in terms of the following dynamical structure factor:
the micelle as a whole [8] Sgan ðQ; oÞ ¼ ½A0 ðQÞdðoÞ þ ð1 A0 ðQÞÞLgan ðQ; oÞ
#LCM ðQ; oÞ: ð2Þ A0(Q) is the elastic incoherent structure factor, EISF, and Lgan(Q,o) is a quasielastic component described by a single Lorentzian term. For the scattering law of the buffer, we adopted a simple phenomenological model already used to describe QENS data from pure supercooled water [9] Sbuf ðQ; oÞ ¼ ½A1 ðQÞL1 ðQ; oÞ þ ð1 A1 ðQÞÞL2 ðQ; oÞ :
ð3Þ
It contains two Lorentzian components: a narrow one, L1, with weight A1(Q), that describes the slow relaxation processes corresponding to the longrange translational diffusion, and a broad one, L2, that accounts for the local dynamics of water molecules. The total resulting scattering law has then been convoluted to the instrumental resolution function. In the fit the diffusion coefficient of the micelles that determines the width of LCM(Q,o) is kept fixed to the value D0=1.8 107 cm2 s1 deduced from the hydrodynamic radius of the micelles. A typical quasielastic spectrum obtained for a 15% ganglioside solution in D2O buffer is shown in Fig. 1. It can be seen that the quasielastic component due to ganglioside hydrogens is much
2 2 Stot ðQ; oÞ ¼ eQ hu i=3 ½Agan Sgan ðQ; oÞ
þ ð1 Agan ÞSbuf ðQ; oÞ þ B:
ð1Þ
A common Debye–Waller factor, given by exp(Q2/u2S)/3 was assumed to take into account fast vibrational motions. Agan is the scattering intensity from gangliosides and from the associated water, and B is a flat instrumental background. The ganglioside contribution is assumed to be described by a confined diffusive motion; it is then convoluted to a Lorentzian LCM(Q,o) that describes the Brownian diffusion of
( 1 for a 15% GM1 Fig. 1. QENS spectrum at Q=1.3 A ganglioside micellar solution in D2O. The total fit as well as the components due to the ganglioside (Iel and IQENS) and to the buffer (continuous line: slow relaxation, and dashed line: fast relaxation) are reported.
ARTICLE IN PRESS P. Brocca et al. / Physica B 350 (2004) e619–e622
narrower than that of the buffer and it can be determined with good accuracy with the adopted instrumental configuration, while the buffer contribution, in the same energy window, is close to a flat background. From a first data fit with free parameters we find Agan=0.4870.1. This value is larger than the one (0.43) expected on the basis of the sample isotopic composition. We attribute this difference to the contribution of water molecules that are closely associated to the micelles, so that their dynamics cannot be distinguished from that of GM1 hydrogens. Agan was then kept fixed to 0.48 in all the subsequent fits. From the Q-dependence of the total scattering intensity we have derived a Debye–Waller-like factor and hence a value of the mean square ( 2 in atomic fluctuation: /u2S/3=(0.0870.02) A reasonable agreement with that measured for other biomolecules [10]. The diffusivity properties of the buffer, not surprisingly, are not identical to those of pure water, and correspond to a slightly reduced mobility. For the confined dynamics of ganglioside hydrogens we have adopted the simple Volino– Dianoux model of a free diffusion within a sphere with rigid walls [11]. The EISF can then be written as 3j1 ðQa Þ 2 Ao ðQÞ ¼ f þ ð1 f Þ ; ð4Þ Qa where j1(Qa) is the first-order spherical Bessel function, a is the radius of the confining sphere and f is the fraction of hydrogen atoms that do not take part to this confined diffusion. The Q-dependence of the EISF and its fit to Eq. (4) is shown in Fig. 2. From the fit we obtain a fraction f of immobile protons f=0.36, and a ( confining radius a=(2.470.2) A. The dynamics of the ganglioside molecule turns out to be quite slow. This is not unexpected, and it can be explained as a consequence of the rather close packing of the bulky sugar headgroups in the hydrophilic shell of the micelle. Also the estimated volume explored by protons is quite shallow ( from the EISF). Indeed it is lower than (R=2.4 A the one that could in principle be accessible, being the area per headgroup at the micelle interface
e621
Fig. 2. Elastic incoherent structure factor derived for the ganglioside contribution. The continuous curve is a fit to a Volino–Dianoux model of confined diffusion inside a rigid sphere.
( 2. One possibility could be that the overall B100 A average dynamics is made up of different contributions, coming from different portions of the molecule. In fact, when dealing with micelles, protons belonging to the hydrophobic core are usually considered as non-diffusing, thus contributing to the elastic part alone of the scattered intensity. This seems not be the case in GM1 micelles. Indeed the value of the fraction f of ‘immobile’ protons from the fit (f=0.36) is too small to account for all the hydrogens in the hydrophobic tails; under this hypothesis one would expect f=0.6. The arrangement of long double tails forced in a non-flat geometry by the hindrance of the headgroups could lead to significant protrusion effects, aided by the fact that the upper region of the chains, close to the headgroup, still contains groups with hydrophilic character. A portion of the hydrophobic moiety could then contribute to the quasielastic part of the scattered radiation, besides the saccharidic one. The resulting measured average dynamics would be lower than that of the headgroups alone. Another possibility is that the dynamics of the headgroup is effectively very slow and more confined than expected. This would lead us to conclude that a remarkably strong coordination occurs among solute and
ARTICLE IN PRESS e622
P. Brocca et al. / Physica B 350 (2004) e619–e622
solvent molecules. Further experiments are underway to clarify this point.
References [1] G. Tettamanti, et al., in: M. Corti, V. Degiorgio (Eds.), Physics of Amphiphiles: Micelles, Vesicles and Microemulsions, North-Holland, Amsterdam, 1985, p. 607. [2] S. Sonnino, et al., Chem. Phys. Lipids 71 (1994) 21.
" M. Corti, E. Del Favero, Physica A [3] M. Boretta, L. Cantu, 236 (1997) 162. " et al., Chem. Phys. Lipids 79 (1996) 137. [4] L. Cantu, " et al., J. Phys. (France) 6 (1996). [5] L. Cantu, [6] P.G. Nyholm, I. Pascher, Biochemistry 32 (1993) 1225. " et al., Physica B 234–236 (1997) 281. [7] L. Cantu, [8] J. Perez, J.-M. Zanotti, D. Durand, Biophys. J. 77 (1999) 454. [9] D. Di Cola, A. Deriu, M. Sampoli, A. Torcini, J. Chem. Phys. 104 (1996) 4223. [10] C. Andreani, et al., Biophys. J. 68 (1995) 2519. [11] F. Volino, A.J. Dianoux, Mol. Phys. 41 (1980) 271.