Thermodynamics of fibrous aggregation of cytochrome c with 1,4-dioxane

Thermochimica Acta 659 (2018) 8–12

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Thermodynamics of fibrous aggregation of cytochrome c with 1,4-dioxane Tomokadu Marutani, Takashi Inomata, Tadashi Kamiyama

⁎

T

Department of Chemistry, School of Science and Engineering, Kindai University, Kowakae 3-4-1, Higashi-Osaka, Osaka, 577-8502, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords: Fibrous aggregation Cytochrome c Dioxane DSC CD

The thermodynamic properties and phase diagram of the thermal-induced fibrous aggregation of cytochrome c with a simple organic co-solvent, 1,4-dioxane, were determined by differential scanning calorimetry, circular dichroism, and scanning electron microscopy at various concentrations of 1,4-dioxane. At x = 0.2–0.3 mol fraction of 1,4-dioxane, cytochrome c transformed to an alpha helix-rich denatured state at 25 °C, which transformed into a fibrous aggregation upon heating. The enthalpy change for the fibrous aggregation, −456 kJ/ mol (x = 0.30), was numerically greater than the thermal denaturation of the native state, 298 kJ/mol (x = 0), indicating that aggregate formation involved a conformation with many intermolecular interactions. At x > 0.3, cytochrome c aggregated and precipitated at 25 °C without heating, and the precipitate transformed into an amorphous state. These results suggest that the previous state is important for transition into the fibrous state. Thermodynamic data on the use of co-solvents for globular proteins will facilitate understanding of the aggregation mechanism.

1. Introduction The conformational state of a protein can be divided into various forms: the folded native state, the unfolded denatured state with loss of tertiary structure and secondary structure which is present in the native state, an intermediate state such as a molten globule state, an aggregated state such as an amorphous aggregation, and an ordered aggregation such as occurs in amyloid diseases. Ordered aggregates have been observed in many proteins and are likely to be one of the basic structural forms of proteins [1–3]. Elucidating the aggregation mechanism is important for preventing and curing protein diseases. The structure of aggregates has been revealed by nuclear magnetic resonance (NMR), X-ray structural analysis, and circular dichroism (CD) [4–10]. However, not much thermodynamic data on the aggregation process exists. J. Kardos et al. [11] and Sasahara et al. [12] reported the kinetic and thermodynamic properties of the aggregation process of β2microglobulin by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). One of the reasons for the lack of such data is the difficulty in quantitatively interpreting the change in spectroscopic intensity because, in general, aggregation inevitably leads to a decrease in protein solubility. Conversely, thermal analysis are effective approaches to compensate for data unavailable by spectroscopy, because thermal methods macroscopically observe whole reactions including aggregated precipitation. Selecting an appropriate condition for aggregating the protein to easily observe the aggregation process is necessary. Generally, one of the ⁎

conditions for inducing aggregation is for the protein to be near its isoelectric point which decreases the electrostatic repulsion between proteins. However, in many cases, aggregation does not result in an ordered form, but an amorphous precipitate. Some proteins containing amino acid substitutions have been reported to form ordered aggregates as amyloids under acidic conditions and/or high pressure [13,14]. However, this method cannot be applied to all proteins. Ultrasonication is one of the effective triggers for aggregation induction [15,16], but it is not easy to detect the thermal information of the aggregation process because the ultrasonic wave creates too large a thermal background. Solvent factors, such as a change in the buffer, can also influence the measured values in thermal measurements and thermal analysis; therefore, it would be desirable to simplify the aggregation conditions as much as possible to accurately determine thermal information on aggregation. This study found that cytochrome c transforms into a fibrous aggregated conformation when heated in the presence of 1,4-dioxane, which enabled us to perform a quantitative thermodynamic analysis. Because this method is simple, not only pathogenic proteins, but globular proteins in general can be used as models for clarifying the aggregation mechanism. The expected increase in the number of aggregation model protein will help clarify the mechanism. Cytochrome c is a good model protein because it is a typical globular and monomeric protein, and there are many reports on its non-native states [17,18], including the fibrous conformation [19,20], although under special conditions. This paper is the first to report thermodynamic data describing the structure in the fibrous aggregation of cytochrome c.

Corresponding author. E-mail address: [email protected] (T. Kamiyama).

https://doi.org/10.1016/j.tca.2017.11.003 Received 4 August 2017; Received in revised form 31 October 2017; Accepted 2 November 2017 Available online 04 November 2017 0040-6031/ © 2017 Elsevier B.V. All rights reserved.

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2. Material and methods

4 2.1. Materials

Exo.

0.30

Bovine heart cytochrome c was purchased from Sigma-Aldrich (95%) and dialyzed with milli-Q water before use. 1,4-dioxane (Kanto Chemical, prime pure grade, 99.9%) was dehydrated using freshly activated grade 4A molecular sieves under a reduced pressure of 0.3 kPa at 327 K. The concentration of cytochrome c as a stock protein solution was determined by absorbance measurement at 280 nm. The sample solutions were prepared by mixing a given weight of the stock protein solution, dioxane, and water. The final concentrations of cytochrome c and the mole fraction of dioxane were determined using dilution factors obtained from the gravimetric and density data for the solvents and solutions. The protein concentration of the solutions was approximately 0.50% and 0.05% w/w. Because the amount of cytochrome c was negligible compared with the amounts of 1,4-dioxane and water, mole fraction of the solution could be regarded as two-component systems of water and 1,4-dioxane.

3 0.25

2

0.20

0.4 0.15 0.2 0.10 0.0 0.05 -0.2 0 -0.4

2.2. Methods

20

30

40

50

60

70

80

90

o

The thermal transition of cytochrome c was monitored with a differential scanning calorimeter [Micro DSC VII evo (SETARAM)] at a scanning rate of 1 K/min. The analysis was performed with a program CALISTO attached to this instrument. All sample solutions and reference solvents were degassed with ThermoVac (Malvern) at least 5 min before DSC measurements. Because the presence of contamination considerably influences protein aggregation, the DSC cell was filled with 6 M urea for 30 min and washed with water to remove any protein contamination after each measurement. DSC curves of cytochrome c were obtained from the difference between the DSC curves for the protein solution and the solvent. Since the DSC cell sealed, the pressure will increase from atmospheric to approximately 0.23 MPa during heating [21]. However, there is almost no influence on the thermal behavior of the protein because the change in pressure is small [22]. CD of cytochrome c was measured using J-720 circular dichroism spectrophotometer (JASCO) with a PTC-348WI temperature control system. The cell length was 1.0 mm and the concentration of cytochrome c was approximately 0.05% w/w. Scanning rate was 1 K/min. All samples were measured within 1 h of preparation. Scanning electron microscope (SEM) images of aggregated cytochrome c obtained after DSC measurements were obtained with an S4800 scanning electron microscope (HITACHI) with an acceleration voltage of 5.0 kV. The aggregations were thoroughly dried under vacuum at room temperature and coated with osmium. Densities of solvents and solutions were measured with a DMA 4500 (Anton Paar, precision ± 10−5 g/cm3) digital density meter calibrated with dry air and double-distilled water. The gravimetric data were obtained using a Sartorius BP210 with a precision of 10−5 g for sample preparation.

Fig. 1. DSC results of cytochrome c in (1 − x) water + x dioxane. The numbers in the figure are the mole fractions of dioxane, x. The dotted lines are integration baselines.

red solution of cytochrome c became colorless accompanied by a red precipitate on heating, indicating that the exothermic reaction was irreversible and most of the cytochrome c aggregated during the first heating. No exothermic peak was observed for mole fractions > 0.4 in the first heating. After heating, different types of precipitates were observed in the sample solution at mole fractions of dioxane ranging between 0.3 and 0.4, as shown in the SEM images in Fig. 2. These results suggest the exothermic peak indicates the irreversible transition from the denatured state to the fibrous state. In general, protein aggregation often occurs during or following denaturation [23,24], because hydrophobic groups exposed by denaturation contribute to intermolecular association due to hydrophobic interaction [25,26]. At the concentration used for the DSC measurement, there are few cases where thermal denaturation and thermal aggregation can be separately observed. Therefore, in most cases, it would be difficult to quantitatively estimate the thermodynamic properties of each transition. However, dioxane significantly separates the transition temperature for denaturation and aggregation, making it possible to determine the change in enthalpy for each transition. The half-height widths of the thermal denaturation and fibrous transition were approximately 10 °C and 4 °C, respectively, indicating that fibrous transition is a very cooperative transition. There was no significant change in the DSC results during a 24-h incubation of the sample at 20 °C following sample preparation, indicating that the state of cytochrome c before heating was independent of time. The averages of the obtained transition temperatures for thermal denaturation, Td, and aggregation, Ta, and the enthalpy changes with standard deviations are listed in Table 1. The enthalpy change for the transition was obtained by dividing the measured heat by the total amount of protein in the system. Ta decreases and the enthalpy change for the aggregation increases, as the mole fraction of dioxane increases. The obtained enthalpy change for the fibrous transition, ΔHa, was −347 to −456 kJ/mol, whereas the enthalpy change for the thermal denaturation, ΔHd, was 298–98 kJ/ mol. Such a numerically large exothermic enthalpy change in the fibrous transition compared with the endothermic thermal denaturation strongly suggests the fibrous conformation has a highly interacted conformation with many intermolecular interactions compared with

3. Results and discussion 3.1. DSC of cytochrome c DSC curves of cytochrome c in aqueous dioxane solutions at various mole fractions of dioxane are shown in Fig. 1. An endothermic peak for thermal denaturation of cytochrome c, which shifted to a lower temperature with increasing mole fraction of dioxane, was observed at 0–0.20 mol fraction of dioxane. The endothermic peak reflects loss of secondary and tertiary structure of cytochrome c for the denaturation. The similar peak was observed for the second consecutive heating indicating the thermal denaturation was reversible. An exothermic peak was observed at mole fractions of dioxane > 0.2. No peak was observed for the second consecutive heating and the 9

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Fig. 2. SEM images of thermal-induced aggregated cytochrome c. (a) dioxane mole fraction, x = 0.30; (b) x = 0.40.

Table 1 Thermodynamic parameters for thermal denaturation and aggregation of cytochrome c in (1 − x) water + x 1,4-dioxane. mole fraction of dioxane

Td °C

0.00a 0.05 0.10 0.15 0.20 0.25 0.30 0.40

83.4 62.9 52.1 43.8 37.5 – – –

± ± ± ± ±

0.1 0.3 0.3 0.5 1.0

ΔHd kJ/mol

Ta °C

ΔHa kJ/mol

298 ± 8 278 ± 7 214 ± 8 153 ± 10 98 ± 18 – – –

– – – – 83.1 ± 0.2 64.9 ± 1.5 53.6 ± 1.2 –

– – – – −347 ± 8 −424 ± 11 −456 ± 9 –

Td – denaturation temperature; Ta – aggregation temperature; ΔHd – enthalpy change for thermal denaturation; ΔHa – enthalpy change for thermal aggregation. Errors are standard deviation of 2–3 measurements. Experimental pressure is 0.10–0.23 MPa. a Comparison with literature value under similar condition (pH 4.6, 40 mM glycine buffer); Td = 78.0, ΔHd = 440 kJ/mol [27].

the monomeric native state. Barone et al. [23] reported the aggregation of bovine serum albumin after thermal denaturation at pH 5.0, observing a large negative enthalpy change for both aggregation (−685 kJ/ mol) and a large positive enthalpy change for denaturation (524 kJ/ mol) at a low protein concentration. Morel et al. [28] showed a specific enthalpy (100 kJ/mol) for fibril disaggregation and unfolding of αspectrin SH3-domain is of a magnitude similar to the enthalpy difference between the partially unfolded species and the fully unfolded state. 3.2. CD of cytochrome c Fig. 3. (a) CD spectra of cytochrome c in (1 − x) water + x 1,4-dioxane. The number beside the thick line is the mole fraction of dioxane, x. (b) [θ]222 of cytochrome c in (1 − x) water + x 1,4-dioxane. The molar ellipticities were calculated with the protein concentration at sample preparation, ≈0.05%.

The enthalpy change in conformational transitions of proteins reflects the difference in protein structure before and after the transition. Therefore, it is necessary to determine the structure not only before but also after the transition. Fig. 3(a) shows the CD spectra of cytochrome c in aqueous 1,4-dioxane solution at various mole fractions and 25 °C. The ellipticity value at 222 nm reflects the amount of secondary structure of the protein [29], which is plotted against the mole fraction of dioxane in Fig. 3(b). The shape and intensity of the spectrum of the protein in water (x = 0) indicated that cytochrome c has a rich α-helix structure in the native state in water. The intensity of ellipticity did not change between dioxane mole fraction from 0 to 0.15. The intensity significantly increased between 0.2 and 0.3, with the spectral shape remaining similar. This increase in intensity indicates an increase in the content of α-helix. It is noted that no endothermic signal of DSC was observed for mole fractions of dioxane > 0.25. The molten globule state, in which the secondary structure exists and the tertiary structure collapses, usually shows an endothermic thermal denaturation [17,18]. The absence of an endothermic transition suggests that the state induced by dioxane is an intermediate state, which is thermodynamically close to the thermally denatured state. At a mole fraction of

dioxane > 0.35, the ellipticity intensity sharply decreased and then fell to zero at a mole fraction of > 0.45, probably because the cytochrome c became insoluble. The strong dependence on dioxane mole fraction is possibly because the dioxane affects the secondary and tertiary structure of the protein. Dioxane induces hydrogen bonding between peptides due to the low relative dielectric constant of dioxane (D = 2.2) [30,31]. Conversely, dioxane may weaken the hydrophobic interactions of the protein due to its lower relative dielectric constant, and the tertiary structure of the protein would therefore be expected to collapse. Our results in other solvents with higher dielectric constants, i.e. dimethyl sulfoxide (D = 47) and 1-propanol (D = 20) in aqueous mixtures, the aggregation temperature of cytochrome c was higher than that in 1,4-dioxane aqueous mixtures and the aggregated conformation was different (data not shown). Dioxane also influences the properties of water. Dioxane has a low dielectric constant, but is soluble in water 10

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state (N) was transformed into the thermal denatured state (DT) upon heating, but not to the fibrous aggregated state (FA) within the measurement temperature range. At 25 °C, the N state transformed into the helix-rich intermediate state (IDX) as the mole fraction of dioxane increased, with the enthalpy change for thermal denaturation gradually decreasing to zero, indicating that the IDX state was thermodynamically close to the DT state. At a mole fraction of > 0.4 at 25 °C, cytochrome c became aggregated and insoluble, as revealed by a decrease in CD intensity, indicating that the aggregate before heating was not in a fibrous form, but in an amorphous aggregated state (AA). At a dioxane mole fraction of 0.2–0.3, the IDX state transformed to the FA state upon heating, which was accompanied by a large exothermic peak. However, this did not occur at a mole fraction of > 0.40, because cytochrome c had been previously transformed to an AA state. Lin et al. [18] showed a phase diagram of the structure of holo-cyto, apo-cyto, and ag-apo-cyto based on added alcohol concentration (TFE and HFIP) and protein concentration, indicating that amorphous and fibrous aggregation occurs in a specific narrow region. These results suggest the previous state of aggregation is important for the transition to the fibrous aggregation, particularly the soluble helix-rich state.

Fig. 4. Temperature dependence of [θ]222 of cytochrome c in aqueous 1,4-dioxane solution. The numbers in the figure are the mole fractions of dioxane. The molar ellipticities were calculated with the protein concentration at sample preparation, ≈0.05%.

4. Conclusion

DT

80

FA

In this study, we determined the thermodynamic properties of the transition to a fibrous form of cytochrome c by DSC following addition of 1,4-dioxane as a co-solvent. The transition to the fibrous form was accompanied by an exothermic reaction that was numerically greater than the endothermic thermal denaturation, thermodynamically suggesting that the fibrous state had a conformation with many intermolecular interactions compared with the monomeric native state. The CD results showed the structure prior to heating is important for the transition to the fibrous state. The Td obtained by the CD measurement was higher than that obtained by the DSC measurement due to the lower protein concentration in the CD measurements. The influence of scanning rate and protein concentration were not included in this study. The accumulation of thermodynamic data on co-solvent induced aggregation will facilitate an understanding of the aggregation mechanism.

60

40

IDX

N 20 0.00

0.05

0.10

0.15

0.20

0.25

AA

0.30

0.35

Fig. 5. Phase diagram of cytochrome c in (1 − x) water + x 1,4-dioxane. The black circles represent Td and Ta. The triangles and open circles represent 25% and 75% of the transition, respectively. N = native state, DT = thermally denatured state, FA = fibrous aggregated state, IDX = helix rich intermediate state, and AA = amorphous aggregated state.

Acknowledgments at any mole fraction. The properties of aqueous dioxane solution are reflected in a negative excess enthalpy [32] and volume [33] at mole fractions of 0.15 and 0.3, respectively, indicating strong interactions between dioxane and water. X-ray diffraction and NMR also show clusters form in the binary solution, which were significantly dependent on the mole fraction [34]. The interaction between 1,4-dioxane and water influences the interaction between water and the protein necessary for the protein to form a native structure, with the conformational transition then occurring at room temperature. Fig. 4 shows the temperature dependence of [θ]222 of cytochrome c at a mole fraction of 0.2, 0.25, 0.3, and 0.4. At a mole fraction of > 0.30, the intensity sharply and irreversibly decreased with heating. After heating, the red protein solution became colorless, and a red precipitate was observed in the sample solution. Therefore, the decrease in intensity indicates a decrease in protein concentration by the aggregation. At a mole fraction of < 0.25, the sharp decrease in intensity was not observed within the measurement range. The obtained midpoint temperature for the aggregation at a mole fraction of 0.30 was approximately 70 °C, which was significantly higher than the 53.6 °C of the DSC result. These results indicate that aggregation is dependent on protein concentration.

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3.3. Phase diagram of cytochrome c Fig. 5 shows the state diagram of cytochrome c based on the transition temperatures, Td and Ta. At a mole fraction of 0–0.15, the native 11

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