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Langmuir 2007, 23, 7118-7126
Effects of Lipid Confinement on Insulin Stability and Amyloid Formation Julia Kraineva, Vytautas Smirnovas, and Roland Winter* Department of Chemistry, Physical Chemistry I-Biophysical Chemistry, UniVersity of Dortmund, Otto-Hahn Strasse 6, D-44227 Dortmund, Germany ReceiVed February 12, 2007. In Final Form: April 17, 2007 We report on a study of insulin incorporation into cubic phases of mono-olein (MO), using synchrotron small-angle X-ray scattering and FT-IR spectroscopy. We studied the thermal stability and aggregation scenario of insulin as a function of protein concentration in the narrow water channels of the cubic lipid matrix and compared it with data for insulin unfolding and fibrillation in bulk water solutions. The concomitant effect of insulin entrapment on the structure and phase behavior of the lipid matrix itself was also examined. We show that the protein’s unfolding behavior and stability are influenced by confinement due to geometrical limitations, and vice versa, the topological properties of the lipid matrix change as well. The addition of insulin already at concentrations as low as 0.1 wt % significantly alters the phase behavior of MO. Surprisingly, new cubic structures are induced by insulin incorporation into the lipid matrix. When insulin begins to partially unfold at higher temperatures, the structure of the new cubic phase changes and finally disappears around 60 °C, where the aggregation process sets in. The aggregation in cubo proceeds much faster and leads to the formation of medium-sized oligomers or clusters, while the formation of large fibrillar agglomerates, as observed for bulk insulin aggregation, is largely prohibited. Hence, the results yield valuable information about the use of cubic mesoporous lipid systems as a medium for long-term storage of insulin and aggregation-prone proteins in general. Furthermore, the results provide new insights into the effects of soft-matter confinement on protein aggregation and fibrillation, a situation usually met in natural cell environments.
Introduction Among lipid bilayer phases, the phases with cubic symmetry are the most complex and intriguing.1-7 Bicontinuous amphiphilic cubic structures are complex viscous fluid networks with a high degree of symmetry and a periodic nanochannel organization. Cubic lipid structures are also involved in different biological processes (e.g., membrane fusion, fat digestion) and occur in cellular and intercellular membranes (e.g., in echinoids, stratum corneum, mitochondria).8,9 Lipidic cubic phases, formed by distinct water and lipid volumes, provide bicontinuous 3-D bilayer matrices that have specific and controllable water channel sizes and large surface areas. With their intertwining periodic networks of aqueous nanochannels of diameters between about 2 and 5 nm, they have proven to be valuable membrane mimetic structures, and they permit nanoencapsulation of drugs and synthesis in confined space.10-12 As proteocubosome carriers, they can serve * Corresponding author. Phone: (49) 231 755 3900. Fax: (49) 231 755 3901. E-mail:
[email protected]. (1) Seddon, J. M. Biochim. Biophys. Acta 1990, 1301, 1-69. (2) Epand, R. M., Ed. Lipid Polymorphism and Membrane Properties, Current Topics in Membranes; Academic Press: New York, 1997; Vol. 44. (3) Conn, C. E.; Ces, O.; Mulet, X.; Finet, S.; Winter, R.; Seddon, J. M.; Templer, R. H. Phys. ReV. Lett. 2006, 96, 108102. (4) Squires, A.; Templer, R. H.; Ces, O.; Gabke, A.; Woenckhaus, J.; Seddon, J. M.; Winter, R. Langmuir 2000, 16, 3578-3582. (5) Kraineva, J.; Nicolini, C.; Thiyagarajan, P.; Kondrashkina, E.; Winter, R. Biochim. Biophys. Acta 2006, 1764, 424-433. (6) Erbes, J.; Czeslik, C.; Hahn, W.; Rapp, G.; Winter, R. Ber.-Bunsen. Phys. Chem. 1994, 98, 1287-1293. (7) Erbes, J.; Winter, R.; Rapp, G. Ber.-Bunsen. Phys. Chem. 1996, 100, 17131722. (8) Deng, Y.; Marko, M.; Buttle, K. F.; Leith, A.; Mierczkowski, M.; Mannella, C. A. J. Struct. Biol. 1999, 127, 231-239. (9) Hyde, S. T.; Andersson, S.; Larsson, K.; Blum, Z.; Landh, T.; Lidin, S.; Ninham, B. W. The Language of Shape. The Role of CurVature in Condensed Matter: Physics, Chemistry, and Biology; Elsevier: Amsterdam, 1997. (10) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. AdV. Drug DeliVery ReV. 2001, 47, 229-250. (11) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4, 449-456.
as carriers of water-soluble recombinant proteins of therapeutic interest, and they can serve as templates to guide the growth of nanostructures and biomolecular scaffolds and for successful crystallization of membrane proteins, which do not easily crystallize in bulk solution.5,13 The structures of the bicontinuous cubic phases (Q) have been described in terms of triply periodic minima surfaces (TPMS), 3-D arrays of connected saddle surfaces with zero mean curvature at every point of the surface. In inverse cubic structures (QII), the lipid monolayer is arranged across either side of the minimal surface, touching it with its terminal methyl groups. This results in 3-D periodic bicontinuous structures formed by distinct water and lipid volumes.14-16 In excess water, 1-mono-oleoyl-rac-glycerol (MO) forms an inversed cubic phase, belonging to space group Pn3m, and it transforms to another cubic phase, belonging to space group Ia3d (with Gyroid minimal surface, Figure 1a), upon dehydration (∼15-40 wt % water) and forms a lamellar LR phase below ∼15 wt % water (at room temperature). A detailed (T,cH2O) phase diagram has been established by Briggs et al.17 Using scattering techniques, the symmetry and structure of the lipid phases can be determined.1,19-21 In this work, we confine insulin molecules into the cubic phases of MO, looking at how the confinement affects the protein stability, unfolding, and aggregation behavior. (12) Nollert, P.; Navarro, J.; Landau, E. M. Methods Enzymol. 2002, 343, 183-199. (13) Rummel, G.; Hardmeyer, A.; Widmer, C.; Chiu, M. L.; Nollert, P.; Locher, K. P.; Pedruzzi, I. I.; Landau, E. M.; Rosenbusch, J. P. J. Struct. Biol. 1998, 121 82-91. (14) Go´z´dz´, W. T.; Holyst, R. Phys. ReV. E 1996, 54, 5012-5027. (15) Schwarz, U. S.; Gompper, G. Phys. ReV. Lett. 1999, 59, 5528-5541. (16) Schwarz, U. S.; Gompper, G. Phys. ReV. Lett. 2000, 85, 1472-1475. (17) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II 1996, 6, 723-751. (18) Harper, P. E.; Gruner, S. M. Eur. Phys. J. E 2000, 2, 217-228. (19) Winter, R.; Ko¨hling, R. J. Phys.: Condens. Matter 2004, 16, 327-352. (20) Winter, R.; Czeslik, C. Z. Kristallografiya 2000, 215, 454-474. (21) Czeslik, C.; Reis, O.; Winter, R.; Rapp, G. Chem. Phys. Lipids 1998, 91, 135-144.
10.1021/la700405y CCC: $37.00 © 2007 American Chemical Society Published on Web 05/25/2007
Lipid Confinement on Insulin Stability/Amyloid Formation
Figure 1. Schematic representation of (a) the cubic phase Ia3d and (b) the dimeric insulin molecule (PDB: 1gui). The bicontinuous cubic Ia3d phase is based on the Gyroid minimal surface and consists of a 3-D network of a curved lipid bilayer that separates two water channels that are mutually intertwined, unconnected, and joined coplanar three × three.
Insulin is a small protein hormone (dimer: 2 × 5.7 kDa, ∼30 Å × 40 Å, Figure 1b) that is crucial for the control of glucose metabolism and in diabetes treatment. It is composed of two peptide chains, A and B, linked together by two disulfide bonds, and an additional disulfide is formed within the A chain. In most species, the A chain consists of 21 amino acids and the B chain of 30 amino acids forming dimers in solution due to hydrogen bonding between the C-termini of the B chains. Insulin is synthesized in the β-cells of the pancreas, where it is stored as a Zn2+ containing hexamer. The biological active form is the monomer. In solution, insulin forms an equilibrium of monomers, dimers, tetramers, and hexamers in its native state, depending on the pH, cosolvents, and concentration.22-24 The monomeric and dimeric forms of insulin are prone to fibrillation, and the insulin aggregation results in the loss of its biological activity. Insulin aggregation resulting in its precipitation is a fundamental obstacle to the development of long-term delivery devices for insulin. Various approaches have been used to prevent aggregation using additives such as surfactants, liposomes, and polymers.25 (22) Adams, M. J.; Blundell, T. L.; Dodson, E. J.; Dodson, G. G.; Vijayan, M.; Baker, E. N.; Harding, M. M.; Hodgkin, D. C.; Rimmer, B.; Sheat, S. Nature (London, U.K.) 1969, 224, 491-495. (23) Whittingham, J. L.; Scott, D. J.; Chance, K.; Wilson, A.; Finch, J.; Brange, J.; Guy, D. G. J. Mol. Biol. 2002, 318, 479-490. (24) Nettleton, E. J.; Tito, P.; Sunde, M.; Bouchard, M.; Dobson, M. C.; Robinson, C. V. Biophys. J. 2000, 79, 1053-1065.
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Insulin is also one of the about 20 known proteins involved in amyloidoses. Amyloidogenic proteins undergo an alternative folding pathway under stressful conditions leading to the formation of fibrils having a cross-β-sheet structure, which is responsible for a number of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, prion diseases, and type II diabetes. Amyloid deposits comprised of fibrillar insulin have been observed both in patients with diabetes26 and in normal aging, as well as after insulin injections.27 Type II diabetes, one of the most common amyloid-related diseases, is characterized by pancreatic amyloid deposits in more than 90% of diabetic patients. These deposits are composed of the islet amyloid polypeptide (IAPP) that is produced in the pancreatic cells and co-secreted with insulin. The early stage of type II diabetes is characterized by insulin resistance, followed by increased insulin and IAPP secretion. Details of the mechanism lying behind insulin aggregation still remain unclear. The aggregation of insulin is accelerated in the presence of denaturants (e.g., urea), whereas stabilizers (sucrose) decrease the aggregation rate.28-30 It has been shown that insulin amyloids’ morphologies and corresponding infrared spectra vary depending on conditions under which the fibrils were grown.31-35 The infrared spectra of the amyloid suggest a parallel arrangement of the β-strands,31 in accordance with a 3-D model proposed for insulin amyloid fibrils.36 Some studies have indicated that insulin aggregation can be triggered by nonspecific interactions with hydrophobic environments, such as Teflon surfaces or air-water or lipid-water interfaces.30,37,38 The main difference between conditions in vitro and in vivo is the presence of a large amount of membrane surface in the latter case, and the formation of amyloid fibrils might be affected by this interaction with lipid surfaces. In this work, we incorporated insulin into a well-defined lipid confinement in the form of cubic mono-olein channels to explore how the confinement modifies the stability and unfolding behavior of insulin as well as the aggregation kinetics and the morphology of the aggregates formed if insulin is destabilized, such as by low pH or higher temperatures. In fact, it has been shown that confinement may lead to significant changes in protein stability and folding rate.39,40 For example, decreasing the size of the cavity may increase the rate of folding until the cavity size becomes only slightly larger than the native state of the protein, at which point (25) Owens, D. R. Nat. ReV. Drug DiscoV. 2002, 1, 529-540. (26) Westermark, P.; Wernstedt, C.; Wilander, E.; Hayden, D. W.; Obrien, T. D.; Johnson, K. H. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 3881-3885. (27) Brange, J.; Andersen, L.; Laursen, E. D.; Meyn, G.; Rasmussen, E. J. Pharm. Sci. 1997, 86, 517-525. (28) Nielsen, L.; Frokjaer, S.; Brange, J.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 8397-8409. (29) Nielsen, L.; Frokjaer, S.; Carpenter, J. F.; Brange, J. J. Pharm. Sci. 2001, 90, 29-37. (30) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 6036-6046. (31) Dzwolak, W.; Ravindra, R.; Lendermann, J.; Winter, R. Biochemistry 2003, 42, 11347-11355. (32) Smirnovas, V.; Winter, R.; Funck, T.; Dzwolak, W. ChemPhysChem 2006, 7, 1046-1049. (33) Grudzielanek, S.; Jansen, R.; Winter, R. J. Mol. Biol. 2005, 351, 879894. (34) Grudzielanek, S.; Smirnovas, V.; Winter, R. J. Mol. Biol. 2006, 356, 497-509. (35) Jansen, R.; Grudzielanek, S.; Dzwolak, W.; Winter, R. J. Mol. Biol. 2004, 338, 203-206. (36) Jimenez, J. L.; Nettleton, E. J.; Bouchard, M.; Robinson, C. V.; Dobson, C. M.; Saibil, H. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9196-9201. (37) Sluzky, V.; Tamada, J. A.; Klibanov, A. M.; Langer, R. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9377-9381. (38) Sharp, J. S.; Forrest, J. A.; Jones, R. A. Biochemistry 2002, 41, 1581015819. (39) Hayer-Hartl, M.; Minton, A. P. Biochemistry 2006, 45, 13356-13360. (40) Ravindra, R.; Zhao, S.; Gies, H.; Winter, R. J. Am. Chem. Soc. 2004, 126, 1224-1225.
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a further decrease in cavity size decreases the folding rate.39 Coupling of FT-IR spectroscopy, small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM) provide an insightful approach for studying conformational and structural changes of the system. The results also yield valuable information about the use of cubic mesoporous lipid systems as a medium for long-term storage of insulin and proteins in general. Furthermore, the results provide new insight into the effects of soft-matter confinement on protein aggregation and fibrillation, a situation usually met in the biological cell. In fact, the data show that the confinement can modify the aggregation kinetics and the morphology of the aggregates formed and vice versa; also, the structure of the lipid environment may be altered. Materials and Methods Sample Preparation. Bovine pancreatic insulin and synthetic mono-olein (1-mono-oleoyl-rac-glycerol, M ) 356.55 g/mol) (MO) was purchased from Sigma Chemical Co. D2O and DCl were purchased from Aldrich. For the FT-IR measurements, insulin was dissolved at a 2 and 4 wt % concentration in D2O. The pD was adjusted to 1.9 with diluted DCl. Clear samples were incubated for 1 h at 20 °C prior to recording first spectra to complete deuterium substitution of solvent-exposed fast-exchanging amide protons. Aqueous MO/insulin samples were prepared by mixing the appropriate amounts of MO and insulin solution (pD 1.9). The level of hydration was kept constant at 20 wt % (∼82 mol %), and the protein concentration was varied from 0 to 4 wt % (corresponding to ∼0.7 protein molecules/unit cell). The samples were subjected to five freeze-thaw cycles (at 25 and -196 °C, respectively) for homogenization of the dispersion. Then, the samples were centrifuged for 5 min at 8000g. The samples obtained were transparent and highly viscous. Throughout all experiments, only fresh samples were used. Small-Angle Synchrotron X-ray Diffraction. X-ray data were collected at the Soft Condensed Matter Beamline (A2) of the DORIS storage ring, Deutsche Synchrotron (DESY), Hamburg, Germany. A camera length of 1 m and 8.05 keV X-rays (λ ) 1.5 Å) were used, and the data were collected as a series of 60 s exposures by using a high sensitivity charge-coupled device detector (MarCCD). Diffraction intensity as a function of reciprocal spacing s (s ) (2/λ) sin θ; λ is the wavelength of radiation, and 2θ is the scattering angle) was obtained by radial integration of the 2-D CCD images using the software FIT2D by A. Hammersley. The incident beam intensity was recorded and used to normalize the individual exposures. Silver stearate was used as a calibrant (d-spacing d001 ) 48.68 Å). From the positions of the low angle Bragg reflections, the mesophase structure and the corresponding lattice constants were determined.19-21 The lattice constant alam of the lamellar phases was calculated from the measured reciprocal spacing slam ) n/alam (order of reflection n ) 1, 2, ...) and the lattice constant acubic of the cubic phases from
shkl ) (1/acubic) xh2+k2+l2, where h, k, l are the Miller indices. The partial specific volume of the lipid (Vl) and water (Vw) components of the system are of similar magnitude (VMO ) 1.062 cm3 g-1, Vw (D2O) ) 0.903 cm3 g-1 at 20 °C; Vp ) 0.74 cm3 g-1) and are assumed to be independent of composition; their temperature dependence is small.41,42 Analysis of Structural Parameters. Under limited hydration conditions of the lipid phase, a number of valuable molecular parameters can be determined from the lattice parameters measured. The internal structural dimensions of the lipid containing phases can be calculated from the measured unit cell dimensions and the sample and water concentration. For cubic phases of the type under discussion, Anderson et al.43 have shown that the molecular cross(41) Kraineva, J.; Narayanan, R.; Kondrashkina, E.; Thiyagarajan, P.; Winter, R. Langmuir 2005, 21, 3559-3571. (42) Bo¨ttner, M.; Ceh, D.; Jakobs, U.; Winter, R. Z. Phys. Chem. 1994, 184, 205-218. (43) Anderson, D. M.; Gruner, S. M.; Leibler, S. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5364-5368.
KraineVa et al. sectional area evaluated on a surface parallel to and at a distance ξ from the minimal surface, and integrated over one of the two monolayers within the unit cell, A(ξ), is related to the experimentally measurable lattice parameter a as follows: A(ξ) ) A0a2 + 2πχξ2
(1)
where A0 is a unitless quantity that describes the ratio of the minimal surface in a unit cell to the quantity (unit cell volume)2/3. The area of the minimal surface in the unit cell is given by a2A0. χ is the so-called Euler-Poincare´ characteristic. The A0 and χ values are defined by the space group and have values of 1.919 and -2, respectively, for the Pn3m phase and 3.091 and -8 for the Ia3d phase.15-18 The area per lipid molecule, Amol, can be obtained by dividing A by the number of lipid molecules in the unit cell, (1 φw)a3/2V, where V is the lipid molecular volume and φw is the volume fraction of water Amol )
2AV (1 - φw)a3
(2)
As in these inverse cubic phases, the sign of the mean curvature is negative (i.e., the lipid headgroup surface bends toward the aqueous phase), and the cross-sectional area per lipid increases from the head to the tail, reducing to zero at the center of the water channel. Eq 2 can therefore be used to estimate the water channel radius in the cubic phases. As at the center of the water channel, where ξ is the sum of the water channel radius rw and the lipid length l, A(ξ) reduces to zero, and one obtains
( )
A0 rw ) 2πχ
1/2
a-l
(3)
Plugging in the values for A0 and χ, we obtain for the water channel radius in the Pn3m phase rw ) 0.391a - l and for the Ia3d phase rw ) 0.248a - l, respectively. The lipid length l of a cubic phase can be calculated by using the lattice parameter a, determined by SAXS, by the known sample composition (i.e., the volume fraction of the lipid, φl (which is 1 - φw - φp, φw being the volume fraction of water and φp the volume fraction of protein), and by using the following relation:
(al ) + 4πχ3 (al )
φl ) 2A0
3
(4)
FT-IR Spectroscopy. The FT-IR spectra were recorded with a MAGNA 550 spectrometer from Nicolet equipped with a MCT (HgCdTe) detector operated at -196 °C. Each spectrum was obtained by co-adding 256 scans at a spectral resolution of 2 cm-1 and was apodized with a Happ-Genzel function. Owing to the large overlap of a H2O IR band with the amide band, D2O was commonly used in infrared spectroscopic studies of proteins. As a background, the D2O/DCl (pD 1.9) spectrum was used. The spectrometer chamber was purged with dry and carbon dioxide-free air. The sample chamber with 4 mm thick CaF2 windows and an optical path length of 50 µm was used. Fourier self-deconvolution of the FT-IR spectra was performed with a resolution enhancement factor of 1.2 and a bandwidth of 15 cm-1. Determination of peak position and curve fitting was performed with OMNIC (Nicolet) and GRAMS (Galactic) software, respectively. The integral intensities of the secondary structure elements of insulin were calculated by analysis of the amide I′ vibration mode of the infrared spectrum. A peak fitting procedure using a mixed Gaussian and Lorentzian peak function allows the overlapping bands to be modeled as the sum of fully resolved ideal peak functions and includes peak picking, baseline fitting, and statistical results.43-48 The amide I′ mode of insulin was fitted in the range of 1696-1595 cm-1 and was analyzed using six (native insulin) (44) Herberhold, H.; Marchal, S.; Lange, R.; Scheyhing, C. H.; Vogel, R. F.; Winter, R. J. Mol. Biol. 2003, 330, 1153-1164. (45) Zein, M.; Winter, R. Phys. Chem. Chem. Phys. 2000, 2, 4545-4551. (46) Panick, G.; Malessa, R.; Winter, R. Biochemistry 1999, 38, 6512-6519.
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Figure 2. Examples of small-angle X-ray scattering patterns and lattice constants a of the lamellar and cubic phases of pure MO (a) and MO/4% insulin (b) in 20 wt % water at pD 1.9 as a function of temperature. or seven (aggregated insulin) mixed Gaussian-Lorentzian functions. From the parameters found, the peak areas (integral intensities) of all components were calculated. The output relative peak areas of the amide I′ band have an approximate error of (2%. Typical R2 values of the peak fitting statistics were in the range of 99.6%. AFM. To separate the insulin aggregate formed within the MO matrix, a 10-fold volume of chloroform was added to the mature MO/insulin samples. The samples were mixed for 5 min and then centrifuged briefly to separate the chloroform and water phase. The chloroform fraction was removed, and a 10-fold volume of deionized water was added. All samples were diluted with deionized water to yield a final 0.001% (w/w) insulin concentration. A total of 20 µL was applied onto freshly cleaved muscovite mica and allowed to dry. The AFM data were acquired in the tapping mode on a MultiMode SPM AFM microscope equipped with a Nanoscope IIIa Controller from Digital Instruments. As AFM probes, Silicon SPM Sensors NCHR (force constant, 42 N/m; length, 125 µm; and resonance frequency, 300 kHz) from Nanosensors were used.49
Results and Discussion SAXS ExperimentssRevealing the Topology and Structure of Phases. SAXS was used to determine the equilibrium phase boundary between the phases and to achieve information about the topology and geometrical parameters of the phases involved. The effect of insulin incorporation on the phase behavior of MO was investigated for MO/water systems with 0.1, 0.25, 0.5, 1, 2, and 4 wt % insulin up to temperatures of 70 °C. For comparison, the phase behavior of the pure MO/water system at pD 1.9 was studied (Figure 2a). At pD 1.9, insulin forms essentially dimers.31 We observed two phase transitions in MO/water: between a lamellar crystalline phase Lc with a lattice parameter a ) 49.3 (47) Cordeiro, Y.; Kraineva, J.; Ravindra, R.; Lima, L. M.; Gomes, M. P.; Foguel, D.; Winter, R.; Silva, J. L. J. Biol. Chem. 2004, 279, 32354-32359. (48) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469-487. (49) Jansen, R.; Dzwolak, W.; Winter, R. Biophys. J. 2005, 88, 1344-1353.
Å and a lamellar liquid-crystalline phase LR (a ) 43.7 Å) at 22 ( 1 °C and a second one between a bicontinuous cubic phase Pn3m (Q224) and a cubic phase Ia3d (Q230) at ∼20 °C. The LR phase disappears at ∼30 °C, and only the Ia3d phase remains (a ) 108.5 Å at 36 °C, da/dT ) -0.2 Å/°C). The phase behavior and lattice constants of MO/water at this low pH are similar to those at neutral pH.41 We calculated the structural parameters of the cubic phase from its unit cell dimensions using eqs 2-4. Upon formation of the Ia3d phase, the water channel radius decreased from 16.7 Å at 20.6 °C to 10.3 Å at 36 °C, and the cross-sectional area at the lipid/water interface decreased from ∼30 to ∼27 Å.2 The effect of temperature on the bicontinuous cubic phase is a decreasing conformational order of the acyl chains, resulting in a decreasing bilayer thickness and an increasing interfacial curvature. The lipid length decreased from 16.6 Å at 36 °C to 15.6 Å at 70 °C, and the water channel radius decreased from 10.3 to 9.6 Å. Addition of insulin already at concentrations as low as 0.1 wt % significantly altered the phase behavior of MO. At low temperatures, all systems showed the first- and second-order reflections of a lamellar (LR) phase at s ≈ 0.022 and 0.044 Å-1, respectively. At higher temperatures, Bragg reflections spaced in the ratio x6:x8:x14:x16:x20:x22 appeared, which are typical for a cubic Ia3d structure. The lattice constant of this cubic phase was nearly the same in all systems: a ) 107.1 ( 1.4 Å at 36 °C (in pure MO/water: a ) 108.5 ( 1 Å) and decreasesed slightly with increasing temperature (aIa3d ) 101.4 Å at 70 °C, da/dT ) -0.1 Å/°C). A similar phase behavior was observed for all MO/insulin systems above ∼0.2 wt % insulin (Figure 3). The lamellar LR phase was stabilized by insulin incorporation, formed instead of an Lc or Pn3m phase, was stable up to 31-35 °C, and transformed into the cubic Ia3d phase at higher temperatures (in the pure lipid system, the Ia3d phase
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Figure 3. Tentative (T,cins) phase diagram of the system MO/insulin in 20 wt % water at pD 1.9.
formed at ∼20 °C and coexisted with the LR phase until ∼30 °C). Cubic phases may be transformed to lamellar phases upon dehydration. Hence, it is possible that the addition of the protein slightly dehydrated the sample within the water channels, thus stabilizing the lamellar structure as observed. The total volume fraction of water in the MO sample did not change drastically, however (φw,total ≈ 0.17). The protein-stabilized LR phase exhibited a lattice parameter a in a similar range as the protein-free LR phase in the binary MO/water system (43-44 Å). A further striking difference in the diffraction patterns of the MO systems with incorporated insulin as compared to the lipid dispersion was the appearance of additional broad peaks at lower scattering angles (clearly visible for insulin concentrations above 0.25 wt %). The intensity of the reflections increased with increasing protein concentration. In MO/4% insulin (Figure 2b), these new peaks appeared at s values of 0.0141, 0.0164, 0.0182, and 0.0198 Å-1, which may tentatively be assigned to two proteininduced cubic lipid structures with low order (probably Pn3m cubic phases X, with main d-spacing of about 70 Å). The water channel radius rw of the Ia3d phase is rather small and decreases slightly with increasing insulin concentration in the solution: for example, from 10.3 ( 0.5 Å for the pure MO/ water system to 9.2 ( 0.5 Å for MO/4% insulin at 36 °C. The insulin dimer has a radius of gyration Rg of 14.9 Å, and the Rg value of monomeric insulin is 11.6 Å, which is very close to that calculated from the crystal structure (11.4 Å).29 Thus, the size of the water channels in Ia3d would just be compatible to the size of the monomer, and the dimers could be easily accommodated only at the three-way junctions of the Ia3d labyrinth structure. Hence, the insulin is probably located within the newly formed cubic structures X only. The Bragg peaks of the cubic phases X vanished at T ) 56.7 ( 2.3 °C in the case of the addition of 0.25% insulin and around 66 °C for 1, 2, and 4% insulin in MO/water (i.e., at temperatures where the dimeric insulin molecules were destabilized and aggregation set in). A tentative (T,cins) phase diagram for all MO/insulin systems measured is displayed in Figure 3. The only phase remaining at higher temperatures was the Ia3d phase. As there was no bulk water coexisting with the cubic phase at these limited hydration conditions, the aggregated protein must be located within the water channels of the Ia3d cubic phase orswhat is more likelyssegregated from the cubic microdomains as separate amorphous-like particles at these high temperatures (vide infra). We then investigated the effect of insulin aggregation on the structure of the lipid matrix as a function of time. The samples with 0.25, 0.5, 1, 2, and 4 wt % insulin were treated for 3 h at
Figure 4. (a) Time dependence of diffraction patterns of MO/4% insulin upon 70 °C treatment. (b) Time dependence of SAXS curves of 4% insulin in MO/20 wt % water upon 70 °C treatment. The curves are background corrected for lipid scattering.
Figure 5. Time dependence of the water volume fraction φw in the cubic Ia3d phase of different MO/insulin samples (20 wt % water) upon 70 °C treatment (estimated error: (0.05).
70 °C, and time-dependent SAXS data were collected. Figure 4a shows characteristic SAXS patterns for MO with 4% embedded insulin at 70 °C as a function of time. The cubic structure was preserved, but the insulin aggregation led to a contraction of the Ia3d cubic lattice, the higher the insulin concentration. The corresponding changes in water volume fraction, as calculated from eq 4, are depicted in Figure 5. Interestingly, the lipid matrix dehydrated upon aggregation of the protein. The water fraction in the Ia3d phase of MO/ 0.25% insulin decreased by ∼10% after 3 h at 70 °C and even by ∼30% in MO with 4% insulin. Upon unfolding and the formation of larger aggregate structures, water was needed for hydration of the exposed amino acid (a.a.) residues. Obviously, no mature fibrillar particles were formed as they hardly contain any water.32,47,49,50,51 To reveal more clearly if the protein exhibited a scattering contribution as well, the MO background was subtracted from (50) Winter, R.; Dzwolak, W. Philos. Trans. R. Soc. London, Ser. A 2005, 363, 537-563. (51) Winter, R.; Dzwolak, W. Cell. Mol. Biol. 2004, 50, 397-417.
Lipid Confinement on Insulin Stability/Amyloid Formation
the SAXS patterns and plotted as a function of time as shown in Figure 4b. Beginning from 10 min after heating to 70 °C, all scattering patterns exhibited a broad scattering peak around Q ) 2πs ) 0.058 Å-1 (corresponding to a real-space dimension d of 108 Å). Accompanied with a steady increase in intensity, the peak position shifted toward slightly smaller momentum transfers, corresponding to larger distances, with time. Very likely, this peak is due to interparticle correlations of small insulin aggregates, with distances d ≈ 7.7/Q. We calculate mean d values of 132 Å after 20 min, 134 Å after 100 min, and 138 Å after 180 min at 70 °C. After cooling back to room temperature, the scattering peak position and its intensity remained unaffected, indicating irreversible aggregation in the confinement. No maturation to larger fibrillar aggregate structures as known from bulk aggregation of insulin seemed to take place.31-34 FT-IR SpectroscopysInsulin Unfolding and β-Sheet Formation. FT-IR spectroscopy was used to follow the secondary structural changes of insulin in confinement and in bulk solution. Information about the secondary structural components can be obtained by analysis of the IR amide I′ band region.43-48 The amide I′ band occurs between 1700 and 1600 cm-1 and represents 76% of the CdO stretching vibration of the amide groups, coupled to the C-N stretching (14%) and C-C-N deformation (10%) modes. The exact wavenumber of the vibrational mode depends on the nature of the hydrogen bonding involving the amide groups, which, in turn, is determined by the particular secondary structure adopted by the protein. Because of the unknown transition dipole moments of the various secondary structure elements, only relative values for the population of conformational states can be given (see Materials and Methods). To obtain detailed information about the secondary structure of insulin in aqueous solution and within the MO channels, we analyzed the amide I′ band of insulin at 2 and 4 wt % in water and of 4 wt % insulin embedded in MO/water in the temperature range from 20 to 70 °C and as a function of time at 70 °C. The FT-IR spectra of insulin at 25 and 70 °C are shown in Figure 6a,b, respectively. In the native state, the band maximum occurs at 1653 cm-1, a position characteristic of proteins with a high R-helical content. At 70 °C, the band shape changes drastically: a prominent band around 1625 cm-1 appears, which is characteristic for the formation of intermolecular parallel β-sheets resulting from aggregation of the unfolded protein.31 Seven bands contribute to the overall amide I′ band area. Table 1 lists the peak positions and relative content of the secondary structure elements for the native insulin at 25 °C, the unfolded insulin at 70 °C, and for aggregated insulin after a 3 h treatment at 70 °C. The structure of the native protein at 25 °C contains 6% intramolecular antiparallel β-sheets, 35% R-helices, 44% disordered structures, and 15% turns. The X-ray diffraction analysis of insulin at pH 2 yields 6% intramolecular antiparallel β-sheets, 41% R-helices, 4% 310 helix, and 49% random structures and turns (PDB entry: 1guj).23 Comparison of the secondary structure of the cubic phase encapsulated insulin with insulin in bulk solution at the same concentration and 25 °C reveals that the protein retains its native secondary structure upon incorporation into the confining lipid matrix. Temperature-dependent FT-IR spectra are shown in Figure 7a for 2% insulin. Partial unfolding and dimer dissociation is indicated by an H/D exchange of the previously unexposed NH groups in the insulin core and concomitant disappearance of the amide II band at ∼1546 cm-1 accompanied by an increase of the amide II′ band at ∼1445 cm-1. This process is completed above 55 °C, in agreement with our previous results.31 The shape of the amide I′ band does not change below ∼60 °C, however,
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Figure 6. Secondary structure analysis of insulin by FT-IR spectroscopy. Curve fitting of the deconvoluted amide I′ band of native insulin at 25 °C (a) and aggregated insulin after 3 h treatment at 70 °C (b) with seven Gaussian-Lorentzian functions: (1 and 7) intermolecular β-sheets; (2) turns; (3) R-helices; (4) disordered structures; and (5 and 6) β-sheets. The insulin concentration was 2 wt % (pD 1.9). Selected results of curve fitting are summarized in Table 1.
except a small red shift of the band maximum by 2 cm-1. Marked changes occur after 15 min at 70 °C, indicated by the increase of the parallel β-sheet aggregation band at ∼1625 cm-1. Notably, curve fitting (Figure 6b and Table 1) reveals that two kinds of parallel β-sheets form during the aggregation process: a β-sheet structure with strong hydrogen bonds (band at ∼1622 cm1) and one with weaker hydrogen bonding pattern (band at ∼1631 cm-1), and the population of a stronger β-sheet structures increases with aggregation time. The more ordered β-sheets are favored in the late aggregated state. The content of R-helices decreases from 35% at 25 °C to 15% after 300 min at 70 °C, and the content of disordered structures decreases from 44 to 14%, respectively. The intermolecular parallel β-sheets form concomitantly at the expense of R-helices and disordered structures. The total content of β-sheets as shown in Figure 7c reaches ∼60% after 300 min and is still slightly increasing with time. At a higher protein concentration (4 wt %), the aggregation process is much faster; it is completed after ∼60 min at 70 °C, where the total content of β-sheets reaches 69% (72% after 180 min). The H/D exchange occurs in a similar way as in the 2% insulin sample and is also finalized at ∼55 °C. The changes in β-sheet and R-helical content upon heating and treatment at 70 °C are compared for all systems in Figure 8. The aggregation process of insulin confined in the MO exhibited a much faster kinetics of intermolecular β-sheet growth. It started at 59 ( 2 °C, immediately after the dimer dissociation. At 70 °C, the content of parallel β-sheets reached ∼49%, while for insulin in bulk solution, we observed only ∼17% parallel β-sheets at this temperature. The aggregation process was completed after ∼60 min, when the parallel β-sheet content reached ∼72%. We observed a similar profile of the aggregation reaction, character-
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Figure 7. Temperature-induced unfolding and aggregation of insulin in solution measured by FT-IR spectroscopy. (a) FT-IR spectra of insulin (2 wt % at pD 1.9) upon gradual heating from 20 to 70 °C at a rate of 20 °C/h. Relative intensity of insulin secondary structure components as a function of temperature (b) and time upon 70 °C treatment (c). The error in determination of the secondary structure elements from the relative peak areas of the amide I′ band from different runs is approximately (2%. Table 1. Vibrational Assignment of IR Bands of the Amide I′ Mode and Relative Contribution (in %) of Each Secondary Structure of Insulin at Different Concentrations and Temperatures (at 25 and 70 °C after Reaching the Temperature and 3 h Later) in MO/Watera structural assignment turns R-helices disordered parallel β-sheets antiparallel β-sheets turns R-helices disordered parallel β-sheets antiparallel β-sheets turns R-helices disordered parallel β-sheets antiparallel β-sheets a
wavenumber (cm-1)
area (%)
wavenumber (cm-1)
2% insulin, 25 °C 1669 14.9 1656 34.9 1640 44.0 1612, 1685 6.2 4% insulin, 25 °C 1667 20.5 1655 34.7 1638 37.7 1611, 1683 7.1 MO/4% insulin, 25 °C 1668 16.8 1655 37.9 1638 38.1 1613, 1683
Estimated error in wavenumbers is (1 cm
7.2 -1
area (%)
2% insulin, 70 °C 1671 16.3 1655 37.3 1639 39.1 1628 0.9 1614, 1681 6.4 4% insulin, 70 °C 1667 19.8 1654 21.2 1640 34.5 1621, 1630 17.4 1612, 1680 7.1 MO/4% insulin, 70 °C 1663 19.6 1653 3.9 1644 17.4 1622, 1632 48.9 1611, 1679 10.2
wavenumber (cm-1)
area (%)
2% insulin, 3 h at 70 °C 1667 10.9 1655 15.0 1643 14.1 1622, 1631 51.2 1614, 1680 8.8 4% insulin, 3 h at 70 °C 1666 7.9 1654 16.5 1640 3.1 1623, 1630 68.1 1614, 1679 4.4 MO/4% insulin, 3 h at 70 °C 1663 10.4 1653 2.3 1645 10.0 1623, 1634 71.7 1611, 1680 5.6
and (2% for peak areas.
ized by conversion of helices and random coil elements to β-sheet aggregates, in fact to two kinds of parallel β-sheets: less ordered strands absorbing around 1632 cm-1 and better packed strands with a stronger hydrogen bonding pattern corresponding to an amide I′ band at lower wavenumbers (∼1623 cm-1). The population of the latter increased with time. Notably, the R-helix content of the protein aggregate seems to be significantly smaller if formed in MO, which may be due to a different topological structure of the aggregate (vide infra). Effect of Insulin Incorporation on the Conformation of the Lipidic Host. The influence of insulin incorporation on the conformational properties of mono-olein was studied by means
of FT-IR spectroscopy as well. The headgroup region of MO consists of three functional groups: two hydroxyl groups (sn-3 and sn-2) and an ester group (sn-1). The stretching vibrations of the terminal hydroxyl group (at ∼1050 cm-1) and the middle hydroxyl group (at ∼1120 cm-1) are sensitive to conformational changes in the headgroup region of the lipid bilayer.52,53 The hydrophobic region of mono-olein consists of one unsaturated fatty acid chain. The chains are packed in an all-trans configuration at low temperatures. A temperature increase enables the rotational (52) Reis, O.; Winter, R. Langmuir 1998, 14, 2903-2909. (53) Reis, O.; Winter, R.; Zerda, T. W. Biochim. Biophys. Acta 1996, 1279, 5-16.
Lipid Confinement on Insulin Stability/Amyloid Formation
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Figure 8. Relative content of R-helices and all β-sheets of insulin in bulk solution (2 and 4 wt %) and of insulin in MO/water (4 wt %) as a function of temperature and time at T ) 70 °C.
energy barrier to be overcome, and gauche conformational states are induced, which leads to characteristic changes of the methylene and methyl vibration frequencies. The wavenumber of the methylene stretching vibration ν˜ (CH2) provides a sensitive qualitative measure of the conformational order and packing of the neighboring acyl chains. We investigated the temperature and time-dependent response of these bands for the MO/2% insulin and MO/4% insulin systems and compared the results with pure MO/water (Figure 9). In the pure MO/2o wt% water dispersion, the wavenumber shifts of the terminal C-OH group of MO appearing around 20 and 30-35 °C clearly indicate the phase transitions Lc + Ia3d f LR + Ia3d and LR + Ia3d f Ia3d, respectively. At low temperatures, the wavenumber of the terminal C-OH group in the system MO/4% insulin was shifted by ∼4 cm-1 to higher values as compared to the pure MO/water system, indicating that the system was in a coexisting lamellar LR phase below ∼35 °C only. Above ∼35 °C, as the cubic Ia3d phase forms from the LR phase, the wavenumber decreased linearly with increasing temperature. As can be clearly seen, the vibrational frequency of the terminal hydroxyl group and its temperature dependence in the cubic Ia3d phase were not markedly influenced by the incorporation of insulin. Upon 70 °C treatment, the wavenumber decreased slightly with time only. The wavenumber of the sn-2 C-OH band of the pure MO dispersion in the ordered Lc decreased from ∼1123 to ∼1121 cm-1 when the disordered LR phase was reached. The LR to Ia3d transition around 30-35 °C was accompanied by a slight change in wavenumber only. At temperatures below ∼20 °C, incorporation of 2 and 4% insulin led to a significant red shift of the sn-2 C-OH band, indicating again that no Lc phase was formed at low temperatures. The addition of insulin also did not markedly change the vibrational frequency of the middle hydroxyl group above ∼35 °C, in accordance with the behavior of the terminal C-OH group. The wavenumber of the symmetric CH2 vibration increased significantly at the Lc + Ia3d f LR + Ia3d transition, from ∼2851 to ∼2854 cm-1. The latter value was observed for the system MO/4% insulin below 20 °C. Evidently, the population of gauche conformers in the MO chains increases at low temperatures upon protein incorporation when compared to the
Figure 9. Influence of insulin incorporation on the conformation of the hydrophilic and hydrophobic regions of MO. Temperature and time dependence of the νs(C-OH sn-3) (a), νs(C-OH sn-2) (b), and νs(CH2) (c) vibrations. For the time-dependent experiments, an equilibration time of 30-40 min was allowed (heating from 20 to 70 °C, where the t is set to zero). The points past the vertical line were taken after cooling to 25 °C.
pure MO/water system, which is due to the formation of the disordered LR phase. The chain packing above ∼20 °C is not significantly influenced by insulin incorporation. All conformational changes of the lipid system upon heating and treatment at 70 °C were reversible.
Conclusion The coupling of FT-IR spectroscopy, the SAXS technique, and AFM proved to provide an insightful approach for studying protein unfolding and aggregation in soft-matter confinement. Embedding insulin in the cubic phase of MO drastically affects its temperature-dependent structure and phase behavior as well as the aggregation kinetics and final aggregate structure of the protein. The structure of the lipid matrix changes upon native insulin incorporation already at low protein concentrations. Insulin incorporation led to the disappearance of the low temperature Lc phase, increased the temperature stability of the lamellar LR phase, and shifted the LR f Ia3d phase transition of MO to slightly higher temperatures. Owing to the geometrical mismatch of protein size and water channel thickness of the lipid matrix, new cubic lipid structures were induced by insulin incorporation into the lipid matrix already at very low concentrations of about 0.1 wt %. The dimeric insulin molecules are probably accommodated within these cubic domains. When insulin began to unfold at higher temperatures, the structure of the new cubic phases changed, and finally disappeared around 60 °C, where the aggregation process set in.
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Figure 10. AFM images of insulin aggregates formed (a) in bulk H2O, pH 1.9 and (b) in cubic MO (80% MO/20% H2O, pH 1.9).
Comparison of the secondary structure of the cubic phase encapsulated insulin with insulin in bulk solution at the same concentration and at 25 °C revealed that the protein retained its native secondary structure. When insulin began to unfold above 60 °C and aggregation began (i.e., after 15 min at 70 °C for a 2% insulin solution), a drastic increase of intermolecular parallel β-sheet formation and a concomitant decrease of R-helical and disordered structures was detected. A more disordered intermolecular β-sheet structure with lower hydrogen bonding strength was (transiently) formed for the higher insulin concentrations. The aggregation in cubo proceeded much faster and led to the formation of smaller aggregates, whereas the formation of large fibrillar aggregate structures, as obtained in bulk solution,49 seemed to be largely prohibited. For example, aggregation of the 4 wt % insulin began at ∼60 °C immediately after dissociation
KraineVa et al.
of the dimers and proceeded a factor of ∼2.5 faster in the confining lipid environment. Upon progressing aggregation at 70 °C, the new cubic phase X hosting the native insulin molecules disappeared, and MO formed an Ia3d structure only. The protein seemed to form small, essentially amorphous and still largely hydrated aggregate clusters, and the protein aggregation led to a drastic dehydration of the Ia3d phase, leading to the shrinkage of the water channel radius. The number of insulin molecules participating in the aggregation process increased with time, but the maximum aggregate size of about 100-140 Å as revealed by SAXS increased only slightly and remained essentially unaltered once the protein had aggregated (i.e., no further maturation of the aggregate structure occurred). A different behavior was observed for aggregation and fibrillation in the bulk solvent (i.e., the soft-matter confinement given by the cubic lipid matrix had a significant effect on the morphology of the aggregate structure). To support these findings, additional AFM measurements were carried out (Figure 10). The AFM images of insulin aggregates formed in bulk water at pH 1.9 show the typical fibrillar structures with diameters of 4-8 nm and lengths of 0.5 µm to several micrometers.49 On the contrary, the protein aggregates grown in the cubic mono-olein phase (80% MO/20% H2O, pH 1.9) essentially consisted of small, amorphous-like mainly 1-2 nm sized particles. Only rarely have protofibrils (of 1-2 nm diameter and up to 500 nm length) been found. As revealed by the FT-IR spectroscopic data, no significant changes in the conformation of the lipid molecules in the fluid phases of the MO matrix were observed upon insulin incorporation and aggregation, neither in the headgroup, nor in the acyl chain region of the lipid, indicating that no significant adsorption at the lipid interface or even incorporation of the insulin molecules into the hydrophobic lipid core took place, even in the partially unfolded and more hydrophobic conformational states. These data thus reveal a very weak coupling between protein and lipid interfacial region, which is essentially of a van der Waals type for this protein only. To conclude, these results yield valuable information about the use of cubic mesoporous lipid systems as a medium for long-term storage of insulin and (aggregation-prone) proteins in general. Furthermore, the data provide new insights into the effects of soft-matter confinement on protein aggregation and fibrillation, a situation usually met in the biological cell. The confinement can modify the aggregation kinetics and pathway and the morphology of the aggregates formed, and vice versa, also the structure of the lipid environment may be altered. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), the EU (Europa¨ischer Fonds fu¨r regionale Entwicklung), and the country North RhineWestfalia. We gratefully thank the staff of the A2 Polymer beamline at HASYLAB/DESY (Hamburg, Germany) for excellent support. LA700405Y
