Secondary Nucleation and Accessible Surface in Insulin Amyloid Fibril

Mar 1, 2008 - Master and Slave Relationship Between Two Types of Self-Propagating Insulin Amyloid Fibrils. Weronika Surmacz-Chwedoruk , Viktoria ...
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J. Phys. Chem. B 2008, 112, 3853-3858

3853

Secondary Nucleation and Accessible Surface in Insulin Amyloid Fibril Formation Vito Fodera` ,†,‡ Fabio Librizzi,*,† Minna Groenning,§ Marco van de Weert,§ and Maurizio Leone†,‡ Dipartimento di Scienze Fisiche e Astronomiche, UniVersita` degli Studi di Palermo, Via Archirafi 36, 90123 Palermo, Italy, CNR - Istituto di Biofisica, U.O. Via U. La Malfa 153, 90146 Palermo, Italy, and Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, UniVersitetsparken 2, DK-2100 Copenhagen Ø, Denmark ReceiVed: October 18, 2007; In Final Form: December 14, 2007

At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical study of the fibrillation kinetics suggests that the early stages of the process are affected by stochastic nucleation events.

1. Introduction Aggregation processes, and in general the physical and chemical instability of proteins, are at the moment a major problem in the development of protein delivery systems. For this reason, a deeper understanding of molecular mechanisms leading to aggregate formation is necessary to improve purification, storage, and delivery of protein-based drugs.1 Moreover, increased knowledge on protein aggregation may clarify some aspects related to several degenerative pathologies like Alzheimer’s and Parkinson’s diseases, type-II diabetes, etc.2-6 A common feature of these pathologies is the presence of ordered protein aggregates in the involved tissue or organ. The ordered aggregates, often referred to as amyloid fibrils, are characterized by a specific local arrangement of cross β-sheet structures. The same structural features have been observed in amyloid fibrils obtained in vitro from a large number of proteins, even many that are not related to any known disease. This indicates that the capability to form amyloid fibrils is a generic property of any polypeptide chain.7,8 The molecular mechanisms responsible for amyloid formation, and protein aggregation in general, are still poorly understood, and many different mechanisms have been proposed. Conformational changes leading to partially destabilized protein structures are commonly recognized as a key step in aggregation processes.6,9-12 After such misfolding, amyloid fibril formation generally proceeds in two steps rationalized in terms of the so-called nucleation-dependent model; the destabilized protein molecules interact with each other, forming a new highenergy species referred to as the nucleus, from which, by * To whom correspondence should be addressed. Phone: +390916234260. Fax: +390916162461. E-mail: [email protected]. † Universita ` degli Studi di Palermo. ‡ CNR - Istituto di Biofisica. § University of Copenhagen.

subsequent addition of protein molecules, amyloid fibrils are formed. In this context, besides the tip-to-tip elongation of the fibrils, other highly cooperative growth mechanisms may take place like branching, fragmentation, and nucleation at the surface of already formed fibrils in solution (secondary nucleation).13-16 In the presence of such mechanisms, the rate of fibril formation depends on the amount of pre-existing fibrils, which gives the process a characteristic exponential growth behavior.14,15 This exponential growth has been observed for several amyloidogenic proteins.17-21 Insulin is a 51-residue protein hormone, with a largely R-helical structure, which plays a crucial role in carbohydrate metabolism. It exists in solution as a mixture of different states (hexamer, dimer, monomer), depending on the environmental conditions.22 In vitro, at low pH and high temperature, insulin is very prone to form amyloid fibrils,23,24 thus constituting a suitable model system to study the molecular mechanisms of amyloid formation.22,25-27 In fact, this model system has recently been used to characterize the mechanical properties of individual amyloid fibrils28 and to determine the growth rate of fibrils by monitoring in real time the increase in their mass.29 The fibrillation process exhibits a strong time dependence, with a pronounced lag phase, followed by a very fast growth of fibrils. This time dependence can be described in terms of the abovementioned secondary nucleation mechanisms,30 in agreement with time lapse atomic force microscopy observations31 and the behavior of insulin fibrillation in confined environments.32 In such a process, a key role is most likely played by the surface of the fibrils,33,34 as previously suggested by Waugh.35,36 In this respect, the autocatalytic nature of insulin fibrillation has also been described by means of a time dependency of the rate constants, to keep into account the growth rate of the activating surfaces.37 The fibrillation-competent species is generally believed to be a partially unfolded monomer, and therefore the

10.1021/jp710131u CCC: $40.75 © 2008 American Chemical Society Published on Web 03/01/2008

3854 J. Phys. Chem. B, Vol. 112, No. 12, 2008 equilibrium between different native species may also affect the overall features of insulin fibrillation.22,25 Here, we present a study on the concentration dependence of human insulin fibrillation in 20% acetic acid (pH 1.8). Under these environmental conditions, insulin is monomeric.22,25,38 Furthermore, it was recently shown by small-angle X-ray scattering measurements that in such conditions, at a concentration of 5 mg/mL, the protein remains monomeric for the duration of the lag phase; afterward, helical oligomers were observed, which may constitute both a structural nucleus and an elongation species in the growth of fibrils.39 In the present work, fibril formation was monitored by Thioflavin T (ThT) fluorescence, a widely used tool for the selective detection of amyloid fibrils.40-42 In aqueous solution and with an excitation wavelength of 450 nm, the fluorescent dye ThT is characterized by a low emission quantum yield (in the 475-600 nm region). In contrast, in the presence of amyloid fibrils a highly fluorescent ThT-fibril complex is formed, allowing the use of ThT as a diagnostic dye for the detection of the presence and rate of formation of amyloid fibrils.40,41 In the last years, the specificity and sensitivity of this dye have been greatly debated, and different mechanisms of the ThTfibril binding have been proposed.43-46 Protofilament, protofibrils, and fibrils are characterized by a high content of cross-β-sheet structures and may form cavities or channels, which are the likely binding location of ThT.43,45,46 The binding is highly directional, with the ThT long axis parallel to the elongation axis of the fibrils.43 Moreover, the cavity size plays a crucial role in determining the characteristics of ThT fluorescence.45,46 Accordingly, an enhanced ThT fluorescence is only possible provided that the surface of the fibrils and the cavities are accessible to ThT molecules coming from the solvent.43 As a general result, this evidence implicates that, for a quantitative detection of fibrils by means of ThT fluorescence, the accessibility of the fibril surface should be taken into account. To examine for undesired effects due to the presence of ThT in solution, we performed experiments at different ThT concentrations, and even in the absence of the dye, by monitoring the fibrillation process by measuring the turbidity of the samples. Moreover, to further characterize the reproducibility of the process by ThT fluorescence, we also performed a statistical study at three different insulin concentrations. 2. Materials and Methods Human insulin was obtained from Novo Nordisk A/S, Copenhagen, Denmark. The zinc content was 0.4% (w/w), corresponding to approximately two Zn2+ ions per insulin hexamer. All samples at different concentrations were singly prepared immediately prior to each experiment. Protein concentration was determined by UV absorbance at 276 nm using an extinction coefficient of 1.0 for 1.0 mg/mL.47 All of the samples used in this study were prepared by dissolving human insulin in 20% acetic acid 0.5 M NaCl (pH 1.8). To avoid formation of salt crystals, the solvent was freshly prepared before each experiment and filtered through 0.22 µm filters (MS 16534, Sartorius), before adding human insulin. Both ThT fluorescence and turbidity experiments were carried out using a plate reader system (Fluostar, BMG Labtech) with 96-microwell polystyrene plates (Nalge Nunc), and each well was filled with 200 µL of solution and four replicates for each sample were measured to determine the reproducibility of the results. For the statistical investigation, at each protein concentration, a protein stock solution (10 mL total volume) containing 20 µM ThT was prepared and then split into aliquots of 200 µL in 44 wells.

Fodera` et al.

Figure 1. Kinetics of human insulin fibrillation in 20% acetic acid, 0.5 M NaCl, 45 °C (λexc ) 450 nm, λem ) 480 nm). The ThT fluorescence is shown as a function of incubation time at different insulin concentrations.

Fibril formation was simultaneously detected on the 44 samples. The plates were incubated at 45 °C without any mechanical shaking of the samples. ThT was purchased as the chloride salt from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). To further purify the product, ThT was recrystallized three times in demineralized water, and the dye concentration in water was estimated by vis absorbance using a molar extinction coefficient of 36 000 M-1 cm-1 at 412 nm.45 For in situ ThT fluorescence, stock solutions of ThT in Milli-Q water were prepared (0.125, 0.25, 0.5, 1, 2, and 4 mM) and stored at 4 °C protected from light to avoid photobleaching. ThT at the desired concentration (from 5 to 80 µM) was added to each well prior to incubating the plate, and the emission intensity at 480 nm was recorded upon excitation at 450 nm. The emission signal was detected from the bottom of the plate every 400 s by an optical fiber system (bottombottom configuration). For fluorescence measurements, the plates were covered with nonsterile Polyolefin sealing tape (Nalge Nunc) to avoid evaporation of the sample. To study the fluorescence intensity dependence on ThT concentration (see Figure 3), the absorption of the excitation beam along the sample volume48 and its effect on the detected fluorescence intensity (Fmeas) have been taken into account for the specific experimental setting (bottom-bottom configuration) by means of the following expression:

Fmeas ∝

I

∫0d c‚I0‚e-c‚‚x dx ) 0(1 - e-c‚‚d)

(1)

where I0 is the intensity of incident light, c is the ThT concentration,  is the extinction coefficient of ThT at 450 nm, and d is the path length of light in the sample. Knowing the values of  and d, the correction factor for the fluorescence data at different ThT concentrations was calculated by evaluating the deviation of eq 1 from a linear concentration dependence:

c‚‚d Fcorr ) Fmeas‚ 1- e-c‚‚d

(2)

No effect is expected from the self-absorption of fluorescence emission, because ThT very weakly absorbs at 480 nm. For turbidity experiments, the optical density at 570 nm was measured during the fibrillation process and the signal was

Secondary Nucleation and Accessible Surface

Figure 2. (A) ThT fluorescence final value (FFV) as a function of protein concentration at three different ThT concentrations. (B) Inverse of the time at which the fluorescence signal reaches 50% of the FFV (1/t50%) as a function of protein concentration at three different ThT concentrations. Error bars represent absolute deviations observed on four replicates.

Figure 3. FFV as a function of ThT concentration for samples at 20 mg/mL protein concentration. (∆) Experimental data and (b) data corrected for the absorption of the excitation light by means of eq 2.

detected from the top of the wells. In this case, the plates were covered with nonsterile advanced Polyolefin sealing tape (Nalge Nunc). 3. Results and Discussion 3.1. Characterization of Fibril Formation. The ThT fluorescence as a function of incubation time is shown in Figure 1 for samples at seven different insulin concentrations in the range 1-20 mg/mL, at 45 °C with a fixed ThT concentration of 20 µM. All kinetics present a characteristic sigmoidal profile

J. Phys. Chem. B, Vol. 112, No. 12, 2008 3855 with a pronounced lag time, in which apparently nothing happens, followed by an abrupt increase of the fluorescence signal. In the whole concentration range, there is no evidence of the biphasic fibrillation behavior observed by Grudzielanek et al. at analogous pH values for bovine insulin, when the protein is dissolved in aqueous solution (without acetic acid).49 In accordance with previous studies,22,25,30 Figure 1 shows that lag time values decrease by increasing insulin concentration, at least in the range 1-5 mg/mL. Analogous fibrillation kinetics were observed with ThT concentrations of 5 and 40 µM (not shown). Two parameters were used to describe the process: (1) ThT fluorescence final value (FFV, see Figure 2A), and (2) the reciprocal of the time necessary to reach 50% of FFV (1/t50%, see Figure 2B). The values obtained for these parameters are shown in Figure 2A and B as a function of protein concentration, for three different concentrations of ThT (5, 20, and 40 µM). Both parameters display a sort of saturation effect for protein concentrations above ∼5 mg/mL. Concerning FFV (Figure 2A), the saturation level clearly depends on the amount of ThT in the sample, but the saturation effect by itself cannot be ascribed to experimental artifacts such as insufficient ThT in solution, at least for ThT concentrations >20 µM. In this respect, we performed a series of measurements at high protein concentration (20 mg/mL) and ThT concentrations spanning from 2.5 to 80 µM. The dependence of FFV on ThT concentration for these measurements is shown in Figure 3; the results (4) display a linear increase of FFV from 2.5 to 10 µM, and then, starting from 20 µM of ThT, the FFV value is almost unaffected by further addition of ThT. It is worthy of note that optical artifacts were also taken into account (see section 2); in particular, the effects of the absorption of the fluorescence excitation beam at different ThT concentrations in the bottom-bottom configuration were estimated (see Methods), and corrected data are also shown in Figure 3 (b). The experimental data are only slightly affected by absorption of the excitation beam, confirming that the attenuation effect at higher ThT concentration has to be related to the peculiarity of fibril-dye binding. Moreover, at the end of the fibrillation kinetics at various concentrations, after centrifugation of the sample, no significant amount of protein in the supernatant was detected (