Effects of high hydrostatic pressure on Rhizopus chinensis lipase: II. Intermediate states during unfolding

Innovative Food Science and Emerging Technologies 45 (2018) 152–160

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Effects of high hydrostatic pressure on Rhizopus chinensis lipase: II. Intermediate states during unfolding

MARK

Gang Chena,b, Ming Miaoa, Bo Jianga, Jian Jinc, Osvaldo H. Campanellaa,d, Biao Fenga,b,⁎ a

State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, PR China School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, PR China c School of Pharmaceutical Sciences, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, PR China d Whistler Center for Carbohydrate Research, Department of Food Science, Purdue University, 745 Agriculture Mall Dr., West Lafayette, IN 47906, USA b

A R T I C L E I N F O

A B S T R A C T

Keywords: High hydrostatic pressure Rhizopus chinensis lipase Native-like state Denaturation Aggregation Molten globule state

High hydrostatic pressure (HHP) is currently considered a well-established technology for processing food and biological materials and there is an interest in investigating the changes in the structural and functional properties of these materials after high pressure treatment. Therefore, the changes in the structure of Rhizopus chinensis lipase (RCL) after high hydrostatic pressure treatment were investigated. Far-UV circular dichroism (CD) spectra showed that the secondary structure of RCL is maintained at pressures below 400 MPa and becomes gradually disordered after higher pressures are applied. Near-UV CD spectra showed that the RCL begins to lose its tertiary structure at pressure over 400 MPa. Fluorescence quenching and the binding of 1-anilinonaphthalene-8-sulfonate confirmed that a partially unfolded intermediate, with loosely compacted conformation and hydrophobic regions, is formed at a pressure of 600 MPa. These results also suggest that RCL maintains a nativelike state at pressures below 400 MPa. Above 500 MPa RCL molecules showed characteristics of being in a molten globule state. Dynamic light scattering (DLS) and atomic force microscopy (AFM) measurements indicated that RCL molecules at these pressures are aggregating. The addition of (NH4)2SO4 to the protein solution could prevent the aggregation, and at 600 MPa the molecule had a hydrodynamic radius approximately 8% larger than that observed for the control sample, which was regarded as being in the molten globule state. The observations suggest that at increasing pressures, the unfolding mechanism of RCL follows well-defined steps from a native state via a native-like structure ending in molten globular state or molecular aggregation.

1. Introduction Proteins are bio-macromolecules with unique conformations and distinctive functional properties. The folded stability of native proteins is crucial to determine many of their functional properties, especially their catalytic ability. The transformation from a folded state to a completely unfolded state in proteins is a complex process involving different possible conformations and paths (Foguel & Silva, 2004). To explain the mechanism of protein unfolding, the classical two-state model was first proposed (Pfeil & Privalov, 1976). However, protein folding was later shown to occur in a stepwise mechanism involving populations of structural intermediates. Recently, it has been confirmed that thermodynamically stable intermediates may exist during the unfolding process (Uversky & Ptitsyn, 1996). In 1983, the concept of molten globule state (MG) was introduced by Ohgushi and Wada (1983). The MG state was then characterized by Kuwajima (1989) as a state in which (1) secondary structure is

⁎

significantly maintained, (2) there is a lack of a precise tertiary interaction due to the tight packing of the side chains to the polypeptide, (3) there is a loosely packed hydrophobic core that increases the hydrophobic surface accessible to the solvent, and (4) the compactness of the protein molecule is characterized by a radius of gyration that is 10–30% larger than that of the protein in the native state. After the conformation of the MG state was found for globular protein, it was reported that under appropriate conditions, many proteins can form a specific, compact and denatured conformation between the molten state and an unfolded state called the pre-molten globule state (PMG) whose features were detailed by Khan, Rahaman, and Ahmad (2011). The features of this state include a less native secondary (about 50%) structure than that of the native state and the absence of a rigid tertiary structure. This state also has a hydrodynamic volume several times larger than that of the protein in the native state and exhibits much weaker 8-anilino-1-naphthalene-sulphonic acid (ANS) binding compared to that observed when the protein is in the MG state (Ohgushi & Wada, 1983).

Corresponding author at: School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, PR China. E-mail address: [email protected] (B. Feng).

http://dx.doi.org/10.1016/j.ifset.2017.08.018 Received 13 February 2017; Received in revised form 30 August 2017; Accepted 30 August 2017 Available online 21 September 2017 1466-8564/ © 2017 Elsevier Ltd. All rights reserved.

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2.3. Lipolytic activity assay

Both MG and PMG are intermediates of partially unfolded states with special characteristics, promoting the further understanding of protein folding. From a thermodynamic standpoint the process promoting the unfolding of proteins involves conformational changes, energy equilibrium and the formation and/or disruption of bonds. Chemical, physical and physiological conditions such as pH (Ahmad, Fatima, Khan, & Khan, 2010), and salts (Record, Guinn, Pegram, & Capp, 2013) are able to change the folding of proteins and to produce relevant intermediate states. The improvement of folding stability by salts and non-electrolytes can prevent aggregation, in contrast with the disruption of protein stability which is mainly associated with the exposure of buried amino acid residues that leads to aggregation under some conditions (Devaraneni, Mishra, & Bhat, 2012). Applications of HHP in biochemistry have been developing in recent decades with applications that transform macromolecular structures, modify of enzyme activity, and alter the metabolism of microorganisms (Marchal et al., 2003; Silva et al., 2014). The pressure-induced unfolding free energy of proteins was found to be distinct from that induced by heat (Torrent et al., 2003). In proteins, high pressure may induce changes that range from small conformational to complete unfolding. The combination of temperature and pressure can also generate enzyme P-T phase graphs (Somkuti, Mártonfalvi, Kellermayer, & Smeller, 2013), which are somewhat different than the P-T phase graphs associated with enzyme stability. Some lipases exhibit higher activity and stability after being treated under HHP, and these changes have been highly correlated with their structural change (Eisenmenger & Reyes-De-Corcuera, 2009). However, to the best of our knowledge, the unfolding path of lipases induced by HHP treatment is seldom studied. Recently, it was observed that the MG state formed during the unfolding of Rhizopus niveus induced by chemical reagents (Rabbani, Ahmad, Zaidi, Fatima, & Khan, 2012). Additionally, HHP treatment has the capacity of inducing the formation of MG state of proteins. On the other hand, it was found that the activity of RCL which is highly homologous to Rhizopus niveus lipase and belongs to globular protein, increased after HHP treatment. Thus in this work, RCL was selected as a model lipase to investigate the process of lipase unfolding caused by HHP. This study is not only relevant because it provides the knowledge of transient conformations of proteins under various high pressure conditions, their conformational information and mechanisms of folding but also because it promotes the use of HHP technology to modify enzymes.

The lipolytic activity was determined using the olive oil-polyvinyl alcohol method (Arima, Liu, & Beppu, 1972; Yang, Chen, Du, Miao, & Feng, 2016). One unit (U) was defined as the quantity of enzyme that liberated 1 μmol of free fatty acid per minute under the assay conditions. All enzyme activity determinations were replicated at least three times. The residual relative activity was calculated by Eq. (1) as:

A A0 (%) =

lipolytic activity after HHP treatment × 100 lipolytic activity before HHP treatment

(1)

2.4. Effect of HHP treatment on RCL catalytic ability The HHP treatment was implemented in a high pressure apparatus (MICRO FOODLAB FPG5740, Stansted Fluid Power Ltd., UK) equipped with a temperature control system. The lipase powder was diluted in 20 mM potassium phosphate buffer (pH 7.5), and the formed solution was placed in a 5 ml plastic tube with a screw lid. The lipase solution was loaded into the chamber of the HHP processing equipment and treated for 10 min. After the treatment, the activity was determined immediately. 2.5. Circular dichroic (CD) measurement The secondary structure changes of RCL after HHP were investigated by CD spectrometry (Bio-Logic MOS-450, France) using a scan rate of 300 nm/min. For the far-UV CD determination the spectra were detected in a cell of 1 mm path length with a protein concentration of 0.1 mg/ml. The near-UV CD spectra were measured with protein concentration of 1 mg/ml in a cell with a 5 mm path length. Each spectrum was the average of three scans. The CD result was expressed as mean residue ellipticity (MRE) (McCabe, Rodger, & Taylor, 2005):

MRE =

θobs 10 × n × C × l

(2)

where θobs is the CD measurement in millidegrees, n is the number of amino acid residues, l is the path length of the cell (cm), and C is the molar concentration of the protein (M). The α-helix content was calculated using MRE at 222 nm (MRE222 nm) according to the following equation (Rabbani et al., 2012):

%(α‐helix) = ⎛ ⎝

2. Experimental

MRE222 nm − 2340 ⎞ × 100 30300 ⎠

(3)

Meanwhile, the β-strand content was estimated using the BeStSel algorithm that reliably distinguishes the features of β-sheets in protein according to the twisting angles between β-strands and that predicts the responsible content by CD spectroscopy and the reference spectra (András et al., 2015).

2.1. Materials Lipase from Rhizopus chinensis CCTCC M201021 was kindly donated by Yiming Biotechnology Company (Taixing, China). 1-Anilinonaphthalene-8-sulfonate (ANS) was purchased from Sigma Chemical Co. (St. Louis, MO). Olive oil, ethanol, polyvinyl alcohol and other chemicals were of analytical grade and purchased from Sinopharm Company (Shanghai, China).

2.6. Fluorescence quenching Fluorescence quenching experiments were performed on a F-7000 spectro-fluorimeter (HITACHI, Japan) at 25 °C with a 1 cm path length cell. In the experiments, aliquots of 2 M quencher stock solution of KI were added to protein solutions (4 μM) to achieve the desired range of quencher concentration (0.1–1 M). To prevent the formation of I3−, the KI stock solutions contained 0.1 M sodium thiosulfate. Excitation was set at 295 nm to excite tryptophan residues only. The excitation and emission slits were set at 2.5 and 5 nm respectively. The emission spectrum was recorded in the range from 300 nm to 400 nm. The decrease in fluorescence intensity at λm was analysed by the Stern-Volmer equation (Eq. (4)):

2.2. Purification of RCL Lipase was purified using the method described by Zhu, Li, Yu, and Xu (2013). The crude lipase was dissolved into 20 mM potassium phosphate buffer at pH 5.5. Then the lipase solution was loaded onto a HiTrap SP FF column (Pharmacia, 5 × 5 ml) and eluted with 0–0.8 M NaCl. Fractions of lipase activities were dialyzed and chromatographed on a HiTrap Phenyl HP (Pharmacia, 5 × 5 ml) column. Lipases were then eluted using the same buffer with an ammonium sulfate concentration gradient from 1.6 to 0 M. Protein fractions with lipases activities were finally dialyzed against phosphate buffer at pH 7.5.

F0 F = 1 + K sv Q 153

(4)

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Table 1. The activity increased at first, reached its highest value at 200 MPa, and then eventually declined as the pressure increased (Table 1). The increase in RCL activity may be attributed to conformational modifications (Chen et al., 2017). It must be noted that the changes in the enzyme activity were small until pressures of 400 MPa, after which very significant effects of pressure were noticeable. The unfolding of enzymes is often associated with their denaturation. At moderate pressure, hydrogen bonds are not sensitive to pressure (Silva et al., 2014), so it could be inferred that the lipase was almost folded and maintained its native-like structure in the pressure range of 0.1–400 MPa. It could also be argued that changes in the enzyme structure were reversible and that the enzyme structure was reverted to its original state after the applied pressure was released. The lipase was obviously deactivated when the pressure was higher and the activity dropped to 89% at 500 MPa. At 600 MPa, the residual activity was 67% of the original value, indicating denaturation of the enzyme to a large extent. However, the enzyme was not completely inactivated. The deactivation of enzymes, which is accompanied by a decrease in catalytic ability, should be attributed to significant changes in conformation, including secondary and tertiary structures, which can be investigated by direct and indirect means (Eisenmenger & Reyes-De-Corcuera, 2009). The measurements were performed at ambient pressure, implying that the protein might have been unfolded under high pressure and could be partially refolded. This condition may also lead to the reduced activity. The results seem to indicate that RCL began to unfold at pressure over 400 MPa.

where F0 and F are the fluorescence intensity of the protein in the absence and presence of the quencher, respectively, Ksv is the SternVolmer quenching constant, and Q is the molar concentration of the quencher. 2.7. ANS binding measurement A stock solution of ANS was prepared in distilled water at a concentration of 4 mM. In the ANS binding experiments, the molar ratio of protein to ANS was 1:20. The excitation wavelength was set at 380 nm and the emission spectra were recorded in the range of 400–600 nm. Both the excitation and emission slits were set at 5 nm. The ANS fluorescence was measured by F-7000 and scanned at least three times at rate of 2400 nm/min. The protein concentration was kept at 0.1 mg/ ml. 2.8. Dynamic light scattering (DLS) analysis DLS measurements were carried out using a DLS device (Zetasizer Nano ZS, Malvern Instruments, U.K.) with a measurement size range of 0.6 nm to 6 μm. All data analysis was performed using the instrument software. Before HHP treatment, all sample solutions were centrifuged for 10 min at 10,000 rpm and then filtered with a filter having an average pore size of 0.22 μm. The sample concentration was kept at 1 mg/ml, the solution was illuminated by a 633 nm laser, and light scattering was detected at an angle of 173°. Each measurement was carried out for every sample with 15 runs. Results were expressed as the intensity or the volume fraction vs. the hydrodynamic radius. The mean hydrodynamic radius (Rh) and polydispersity were estimated on the basis of an autocorrelation analysis of the scattered light intensity data based on the translational diffusion coefficient (D) given by the Stokes–Einstein equation:

Rh =

kT 6πηD

3.2. Secondary structure of RCL investigated by far-UV CD The effects of HHP treatment on the secondary structure of RCL was investigated by far-UV CD spectroscopy. As shown in Fig. 1A, the spectrum of RCL at 0.1 MPa can be characterized by three negative peaks: two peaks at 208 and 222 nm representing an α-helix structure and one at 217 nm representing a β-sheet structure in RCL. The CD spectra of RCL treated for pressures below 400 MPa were highly overlapping whereas the spectrum corresponding to a pressure of 500 MPa starts to be distinctive and shows some differences, which become significant and noticeable when RCL is treated at pressures of 700 MPa (Fig. 1A). The results suggest that RCL did not undergo denaturation at pressures below 400 MPa, which is consistent with the idea that the secondary structure of the protein is not highly sensitive to low and moderate pressures (Silva et al., 2014). However, values of MRE at the minimum wavelengths of 208 and 222 nm increased at pressures over 500 MPa, especially at 700 MPa, suggesting a decrease in the α-helix. Likewise, the β-strand content fluctuated at approximately 25% for pressure of 0.1–400 MPa and began to increase at 400 MPa. In particular, the content increased to 33% at 600 MPa and 42% at 700 MPa (Table 1). The loss of α-helix content may contribute to the significant increase in β-strands via conformational rearrangement. The high pressure treatment can also cause the permanent loss of secondary structure by disrupting hydrogen bonding, just as temperature and chemicals agents do (Jung, Savin, Pouzot, Schmitt, & Mezzenga, 2008). The trend of the changes in MRE222 nm with pressure can be seen more clearly in Fig. 1B. The MRE222 nm of RCL remained stable at pressures below 500 MPa and began to increase remarkably thereafter. Thus, based on these changes in MRE222 nm with pressure, two phases could be defined. In the first phase, from pressures of 0.1 to 400 MPa, there is no significant change in the MRE222 nm values whereas in the second phase (pressure range of 500–700 MPa), a remarkable increase in MRE222 nm can be observed. This finding suggests that the secondary structure of RCL is permanently altered at pressures over 400 MPa. Therefore, the 400 MPa pressure may be considered the pressure at which unfolding begins. The transition pressure or transition zone has different values depending on the protein. For example, β-lactoglobulin partially unfolds at pressures between 150 and 300 MPa (Funtenberger, Dumay, & Cheftel, 1997) and completely unfolds at pressures near

(5)

where Rh is the hydrodynamic radius, k is Boltzmann's constant, T is the absolute temperature, η is the viscosity of the solvent (water in this case), and D is the translational diffusion coefficient. 2.9. Atomic force microscopy (AFM) morphological analysis AFM measurements were performed on a CSPM 5500 scanning probe microscope (SPM) platform (Being Nano-Instruments, China). Pure RCL solutions (1 mg/ml) were treated at different pressures (0.1, 400, 500, and 600 MPa) for 10 min at 40 °C. The treated samples were properly diluted and pipetted on clean mica plates using droplets of approximately 10 μl for lipase deposition and then kept in a pre-marked petri dish to dry. Finally, the samples were imaged using the tapping mode with a silicon PPP-NCHR AFM probe (thickness 4.0 ± 1 μm, length 125 ± 10 μm, height 10–15 μm). 3. Results and discussions 3.1. Effect of HHP on RCL activity Proteins are easily affected by various energetic and topological interactions, which are related to their functions. High pressure-induced changes in enzyme conformation are often accompanied by changes in the catalytic ability of enzymes (Chen, Peng, Zhang, & Yan, 2013). Thus, enzyme activity is a sensitive indicator of changes in the conformation of enzymes that are treated under high hydrostatic pressures. The activity and the fluorescence intensity of RCL treated at different time were measured (Fig. S4). The results showed that both were stable after 10 min. Thus treatment time was set at 10 min. The relative residual activity of RCL after HHP treatment is given in 154

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Table 1 Pressure dependence of the enzyme activity, α-helix content, β-strand content and quenching parameters, hydrodynamic radii (Rh) and polydispersity (PDI). Pressure (MPa)

0.1

200

400

500

600

700

A/A0 (%) α-Helix (%) β-strand (%) Ksv f Rh1 (nm) % PDI1 Rh2 (nm) % PDI2

100.0 ± 1.0 45.6 25.0 10.37 ± 0.52 0.31 ± 0.02 3.73 30.7 3.82 30.3

116.5 ± 1.5 44.3 25.1 10.44 ± 0.27 0.33 ± 0.01 3.62 30.3 3.80 30.5

98 ± 0.8 45.1 25.5 10.37 ± 0.61 0.30 ± 0.01 3.72 42.6 3.79 33.4

89 ± 1.3 40.5 27.5 11.12 ± 48 0.38 ± 0.01 3.73 42.0 3.81 29.8

67 ± 2 38.2 33.0 12.52 ± 0.53 0.29 ± 0.02 128.20 18.9 4.02 51.9

– 24.2 42.0 – – 146.90 19.5 4.07 54.5

Fig. 2. Near-UVCD spectra of RCL after HHP treatment.

previous observation that the intrinsic fluorescence of RCL barely changed at pressures below 400 MPa. When the pressure was elevated continuously to 500 MPa, the spectrum remained the similar shape but had a significantly different value, indicating that tertiary structure changed. This assumption was supported by the intrinsic fluorescence at 500 MPa where the fluorescence intensity began to increase significantly with a shift in the maximum wavelength (Fig. S1). Notably, when the pressure was increased to 600 MPa, the spectrum exhibited a broad band (280–300 nm) with little fine structure, suggesting that the aromatics were in several conformations (Woody & Dunker, 1996). Moreover, the peak at 250–260 nm generated by SeS bond and phenylalanine gradually decreased with pressure. This loss in the nearUV CD signal corresponds to the contributions of the partially unfolded state of RCL. A similar observation has also been found in other lipases treated with extreme acid or alkali pH conditions (Ahmad et al., 2010). Higher pressure produces changes in the distribution of the side chains of aromatic residues and their environment. Furthermore, high pressure enhances the transition that results in deprotonation to form charged residues (Randolph, Seefeldt, & Carpenter, 2002). All these changes often accompany the unfolding of the protein. In consideration of the change in intrinsic fluorescence, this observation suggests that the use of HHP at 600 MPa led to a marked loss of tertiary structure in RCL.

Fig. 1. Secondary structure of RCL determined by far-UV CD. (A) Spectra as function of pressure. (B) Effect of pressure on MRE222 nm.

1000 MPa (Hayakawa, Linko, & Linko, 1996). The remarkable loss of αhelix content observed at high pressure (Fig. 1A) would imply that the protein unfolding could be accompanied by a change in hydration due to the loss of hydrogen bonds. Thus, the pressure-changed unfolded structure of the protein would suggest that the solvation and distribution of water-excluded cavities could be changed by pressure (Silva et al., 2014; Silva, Foguel, & Royer, 2001). All of these changes influence the transient state of RCL between the folded and completely unfolded states.

3.4. Quenching investigated by intrinsic fluorescence 3.3. Effect of high pressure observed by near- UV CD The exposure of tryptophan residues is an indicator of the lipase conformation related to its tertiary structure, so fluorescence quenching was examined using KI as an ionic quencher. When the relative fluorescence intensity was plotted against the concentration of KI, the relationship was not linear as predicted by the Stern-Volmer equation (Fig. 3A). A similar phenomenon was observed in the quenching of lectin (Sultan, Rao, Nadimpalli, & Swamy, 2006) and wheat NADPmalic enzyme (Spampinato, Ferreyra, & Andreo, 2007). This finding

To better understand the nature of the intermediate states induced by HHP, alterations in the lipase tertiary structure were monitored using near-UV CD, and the results are shown in Fig. 2. The near-UV CD spectra of RCL at pressure from 0.1 to 400 MPa show a similar shape without significant characteristic peaks such as positive peaks around 275 and 295 nm. These spectra at pressures below 400 MPa suggest that the tertiary structure changes were small, agreeing with our 155

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A

1200

F at 480 nm

1000 800 600 400 200 0 0

100

200

600

700

B

1200 Fluorescence intensity

300 400 500 Pressure (MPa)

0.1 MPa 400 MPa

1000

500 MPa

800

600 MPa

600

650 MPa

400 200 0 400

440 480 520 560 Emission wavelength (nm) C

90 Fluorescence intensity

Fig. 3. Quenching of the Trp fluorescence of RCL by KI. (A) Quenching curve at atmospheric pressure. (B) Modified Stern-Volmer plots at different pressures.

was probably due to the presence of at least two fluorophores in this lipase with different accessibilities to the quencher (Lakowicz, 2013). Consequently, the accessible fluorophores and the effective quenching constant (Ksv) were calculated by a modified Stern-Volmer equation (Eq. (6)):

F0 ΔF = 1 f + 1 (f K sv c )

(6)

600

0.1 MPa 200 MPa

75

400 MPa

60

500 MPa

45 30

15 0

where ΔF (=F0 − F) is the change in fluorescence intensity at any point in the quenching titration, and f is the fraction of the total fluorophores accessible to the quencher, c is the concentration of quencher. Ksv is an important parameter for estimating the exposure of aromatic residues and evaluating the compactness of the protein molecules (Ahmad et al., 2010). Based on Eq. (6), a linear regression can be used to obtain the value of f and Ksv for different quenching conditions (Fig. 3B). The results of that regression are reported in Table 1. As expected, f and Ksv fluctuated at pressures below 400 MPa because of small conformational changes whereas at pressures over 400 MPa the value of Ksv increased gradually. Under higher pressure, the increase in fluorescence intensity can be induced by the by the movement of the quenchers surrounding Trp residues (Lakowicz, 2013). Upon unfolding, the protein becomes loosely compacted, and the Trp residues are easily quenched, resulting in higher values of Ksv after HHP treatment at pressure over 500 MPa. This finding may be attributed to the fact that the Trp residues interacted more easily with I− after the treatment. On the other hand, the value of f remained almost unchanged until 400 MPa. Then the value of f increased almost by one third at 500 MPa but dropped again at 600 MPa. Other researchers have pointed out that the redistribution of charged groups might also influence the accessibility of Trp residues (Komath & Swamy, 1999; Sultan et al., 2006). High pressure processing

400

440

480 520 560 Emission wavelength (nm)

600

Fig. 4. ANS binding to RCL treated at various pressures. (A) Change in extrinsic fluorescence intensity measured at 480 nm. (B) Extrinsic fluorescence spectra under various pressures. (C) Fluorescence spectra of the ANS solution plotted versus pressure (control).

changes the electrostatic interaction of the protein (Weber & Drickamer, 1983) and might contribute to the observed fluorescence quenching results. The low accessibility observed with I− at 600 MPa might be due to the presence of charged residues in the vicinity of some of the tryptophan residues (Sultan et al., 2006). Thus, the high value of Ksv can be regarded as an index of the loosely compacted conformation of the protein. The results of the quenching experiments are in good agreement with the observations obtained in the CD studies. 3.5. ANS binding to pure RCL ANS is an extrinsic fluorescence probe, which preferentially binds to a loosely packed solvent-accessible hydrophobic core. It is frequently used to study the exposure of hydrophobic clusters on protein surfaces, which is observed by the increase in the fluorescence intensity at 156

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RCL. Two peaks, at 3.7 and 105 nm are noted. The larger size indicates the presence of soluble aggregate particles. The soluble aggregates are formed during the purification of the enzyme and the pretreatment could not remove them completely. In the following discussion, the hydraulic radius of RCL in the absence of salt is designated as Rh1, whereas the hydraulic radius of RCL in the presence of salt is Rh2. The same labeling is also used to distinguish the polydispersity index (PDI) as PDI1 and PDI2 when salt is absent and present, respectively. To avoid the shielding of the laser light beam from the aggregates, the size distribution was expressed as the volume percentage. As discussed in previous sections, hydrophobic cavity changes in protein hydration are promoted by high pressure conditions, which appears to control the hydraulic radius (Rh) of the particles (Halle & Davidovic, 2003). In Table 1, Rh1 (in absence of salt) remained approximately constant and stable until pressures of approximately 500 MPa. However, Rh1 exhibited a marked jump when the pressure increased from 500 to 600 MPa, indicating the formation of new aggregates after HHP treatment. For example, it was observed that the presence of a peak with a radius of 128 nm after the HHP treatment at 600 MPa (Fig. 5B). The polydispersity index (PDI) for all samples treated below 600 MPa was over 20%, which suggests that the samples were not monodisperse, i.e., soluble aggregates probably existed before HHP treatment. The PDI1 was below 20% for RCL subjected to pressures over 600 MPa, indicating a more uniform size distribution. The shift of the peak from 3.73 nm for the pressure of 0.1 MPa to approximately 120 nm for pressures over 500 MPa (Table 1) indicates that a large portion of the protein present formed aggregates after the HHP treatment. The CD and fluorescence spectra studies indicated that at pressures over 500 MPa, RCL was partially unfolded with a significant increase in the hydrophobic regions after HHP treatment, which appears to have promoted intermolecular hydrophobic interactions (Amin, Barnett, Pathak, Roberts, & Sarangapani, 2014; Randolph et al., 2002). The protein was unfolded by high pressure. Once the pressure was released, the protein failed to refold to the correct native state but formed a partially unfolded intermediate prone to aggregation (Foguel & Silva, 2004; Randolph et al., 2002). Another factor is that the pre-existing soluble aggregates (Fig. 5A) might have enhanced aggregation due to a “seeding effect” (Gregory, Vladimir, Bruce, Alexander, & Christopher, 2016). In Table 1, Rh2 was determined with the addition of 0.1 M (NH4)2SO4. When the pressure was below 500 MPa, the presence of salt did not significantly affect the hydrodynamic radius. However, at 600 MPa the addition of salt inhibited the aggregation of protein and resulted in a less homogenous distribution. (NH4)2SO4 can prevent aggregates by changing the structure of the water and the hydrogen bonding in the protein molecule (Hamada, Arakawa, & Shiraki, 2009). The use of salt has been employed to prevent the heat-induced aggregation of lysozyme (Hirano et al., 2007). With the unfolding of RCL, a small amount of the salt may enter the interior of RCL, thereby disrupting the hydration of hydrophobic groups and hydrophobic interactions likely preventing protein aggregation (Chi, Krishnan, Randolph, & Carpenter, 2003). Thus, the results seem to indicate that salt prevents the aggregation of RCL by affecting the hydration of the protein, the distribution of charge on the protein surface and the hydrogen bonding. However, the effect of salt on the unfolding stability and aggregation of proteins varies with the concentration and type of salt used. The result indicates that the introduction of the salt changes the hydrodynamic radius of RCL subjected to pressure of 600 MPa. Table 1 shows that HHP treatment at 600 MPa in the presence of salt increases the hydrodynamic radius of treated RCL by approximately 8% when compared to that of native RCL. In combination with the change in intrinsic fluorescence (Fig. S2) and ANS fluorescence (Fig. S3), the Rh2 at RCL treated at pressures of 600 MPa suggests the formation of a MG state.

480 nm (Miroliaei, Ranjbar, Naderi-Manesh, & Nemat-Gorgani, 2007). ANS can also be used to detect non-native, partially folded conformations of globular proteins (Rodionova, Semisotnov, Kutyshenko, Uverskiĭ, & Bolotina, 1989). Fig. 4A shows the fluorescence intensity at 480 nm as a function of the applied pressure in the HHP treatment, whereas Fig. 4B contains the spectra obtained for the RCL-ANS complex. However, the fluorescence of ANS was rarely affected by HHP treatment (Fig. 4C). The native RCL showed weak fluorescence intensity at 480 nm due to the lack of significant ANS binding sites. The binding of ANS to hydrophobic regions increases the fluorescence intensity, and within the pressure range of 0.1–400 MPa the fluorescence intensity remained stable and very low. However, a marked increase in the fluorescence intensity was observed starting at pressures of 400 MPa (Fig. 4A). The spectra also showed a large shift from 515 nm to 480 nm (Fig. 4B) at pressure higher than 500 MPa, indicating the presence of hydrophobic cores. This observation supports the hypothesis that high pressures make the protein more solvated and increase the accessibility of hydrophobic regions to the solvent (Silva et al., 2014; Weber et al., 1992). Additionally, the result indicate that a significant structural change corresponding to the of RCL transformation from the native state to a partial unfolded conformation is promoted by the high pressure conditions. The results also suggest that unfolding begins at approximately 500 MPa. The fluorescence change confirms the assertion that partially folded intermediates are formed after high pressure (500–600 MPa) treatment, which is confirmed by the UV and CD studies. The results also indicate the presence of a loosely compact hydrophobic core able to increase the hydrophobic surface accessible to the solvent at high pressures. This finding may suggest the existence of the MG state. In addition, the turning point pressure to reach a MG state varies with the type of protein. For, instance another study has been reported that the apomyoglobin protein transitions to a MG state at pressures approximately 200 MPa (Kitahara, Yamada, Akasaka, & Wright, 2002). 3.6. DLS measurements DLS was used to measure the hydrodynamic radii of RCL in the different states reached after the HHP treatment. Fig. 5 and Table 1 summarize the DLS results. Fig. 5 A shows the size distribution of native

Fig. 5. Measurement of the hydrodynamic radius of RCL subjected to different pressures (A) Intensity vs. hydrodynamic radius at 0.1 MPa. (B) Volume fraction vs. hydrodynamic radius at 600 MPa.

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Fig. 6. Topology of RCL samples treated with different pressures. (A), (B), (C), and (D) correspond to pressures 0.1, 400, 500 and 600 MPa, respectively, in the absence of (NH4)2SO4, whereas (E) corresponds to the 600 MPa pressure in the presence of (NH4)2SO4. For clear observation, (F) and (G) are the 3D pictures of (D) and (E), respectively.

treatment of RCL in the presence of salt did not lead to aggregates even at pressures as high as 600 MPa and the resulting RCL had similar sizes as the native RCL (Fig. 6E). Together with the DLS and CD analysis, the AFM measurement confirms that RCL aggregation is promoted during the process of unfolding, further supporting the hypothesis that RCL aggregation may begin in a partially unfolded state of the protein (Foguel & Silva, 2004).

3.7. AFM analysis AFM analysis is widely used in the topological characterization of the surface of biological systems. AFM is also a useful tool to evaluate aggregation of proteins (Baron et al., 2005). As discussed in previous sections, HHP treatment can influence the aggregation of enzymes through structural modification. To confirm whether HHP is able to induce RCL aggregation, an AFM analysis was employed to visualize the appearance of RCL after HHP treatment at different pressures. The images, which provided a molecular resolution, of RCL treated at different pressures are illustrated in Fig. 6. To visualize a single particle of native RCL treated at 0.1 MPa, a lower concentration was used; therefore, the number particles observed in Fig. 6A is obviously lower than those observed in the images of samples treated at high pressures. As illustrated in Fig. 6, there is little variation in the size of the particles, which are seen as single separate entities with no aggregation for samples treated at pressures 0.1, 400 and 500 MPa (Fig. 6A–C). They all have approximately a circular shape similar to many proteins in a nonaggregate state with about 30-nm relative height. RCL in the absence of salt shows some aggregation in different clusters after pressures treatment at 600 MPa despite the small number of single separate entities (Fig. 6D). Some aggregates adopt an elliptical shape of a larger size (Fig. 6F), and others cluster together without developing a definite shape with a relative height of 500 nm. The

3.8. Process of unfolding induced by HHP treatment The results obtained in the present research appear to indicate that the unfolding of RCL induced by high pressure treatment could follow a stepwise mechanism, which is schematically illustrated in Fig. 7. In the pressure range from 100 to 400 MPa, the secondary structure of the protein is mostly maintained, the tertiary structure is not disrupted significantly, and a few hydrophobic regions are exposed to the solvent. The CD and fluorescence spectra revealed only small variations in the RCL conformation upon treatment at pressures in the range of 100–400 MPa compared to the conformation of RCL in the native state. The small difference between the native-like state (NL) and the native state may be due to local unfolding and conformational rearrangements (Neumaier & Kiefhaber, 2014). It is likely that those small changes are responsible for the change in the activity of RCL when treated at those pressures. 158

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Fig. 7. Mechanistic process of RCL unfolding induced by HHP treatment under various pressure conditions that was elucidated in the present research. The blue dots in the figure represent water molecules. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

conformational rearrangement of RCL, further contributing to the aggregation process after depressurization. In addition, from other results concerning secondary and tertiary structure, the exposed hydrophobic area and the hydrodynamic radius were in agreement with the characteristics of a MG state. In combination with the observations in RCL samples treated at 600 MPa, it could be inferred that the protein should be in a MG state before aggregating at 600 MPa. Salt played an important role in the unfolding of RCL. The presence of salt effectively prevented aggregation. The results of ANS measurement binding and intrinsic fluorescence (cf. supporting information) were similar to that described previously, suggesting the features of a MG state and suggesting that the salt did not affect the process of unfolding. Marchal et al. (2003) reached similar conclusions on the unfolding of human Ataxin-3 at pressures in the range of 300–650 MPa, where the protein was not affected in the presence of NaCl. In the present study, an AFM approach showed that in the presence of the 0.1-M salt, no aggregation was observed at 600 MPa (Fig. 6E) and the hydrodynamic radius was approximately 8% larger than that of the native RCL (Table 1), which confirms that the addition of salt prevents aggregation.

As suggested by previous research, changes in the microenvironment can cause dynamic NL ensemble of protein conformations with small conformational shifts (Beauchamp, McGibbon, Lin, & Pande, 2012; Meuzelaar et al., 2013), which lays the foundation for the generation of the NL state. The presence of an NL state in high-pressure folding/unfolding studies has been observed for staphylococcal nuclease (Vidugiris, Markley, & Royer, 1995) and ribonuclease (Panick & Winter, 2000). Based on the literature, it could be inferred that RCL first transforms from the native state into an NL state at which the enzyme is able to maintain or even increase its catalytic ability, with some noticeable fluctuations in the measured RCL activity. This assumption is also supported by the evidence that native proteins can exist in several alternatively packed, compact folded states, which may affect their functions (Neumaier & Kiefhaber, 2014). A pressure of 400 MPa appears to be the turning point above which pressure-induced unfolding becomes more significant. However, this native-like transition state was somewhat different than the NL state reported previously. For example, it was found that modifications of a loop region in acylphosphatase lead to only small variations in the protein, indicating a native-like state (Dagan et al., 2013). However, these modifications, increase the thermodynamic stability of the enzyme by increasing the folded state entropy (conformational entropy), thus strongly compromising enzymatic activity. With increase in pressure up to 600 MPa, the protein underwent a pronounced unfolding with marked loss of ordered structure, its secondary structure was disrupted, and some α-helix structure changed into a disordered structure; however, the amount was still limited. Nevertheless, the tertiary structure was disturbed. The protein was less compact and more hydrophobic regions were exposed to the solvent. All of these features suggested that at these higher pressures a MG state might be achieved. However, measurements of the hydrodynamic radius by DLS and AFM indicated that aggregation also occurred after treatment at 600 MPa. While, the transition point, observed at 500 MPa, can be regarded as an intermediate state where no aggregation is observed. The intermolecular disulphide bond is one factor contributing to the aggregation of proteins and high pressure is capable of inducing the formation of new disulphide bonds (Wang et al., 2008). However, measurements indicated that disulphide bonds were not modified (cf. supporting information). One possible explanation is that the high pressure condition caused the protein to exhibit more hydrophobic regions and forced water molecules to penetrate the protein molecule (Chi et al., 2003). The folding stability was thus disturbed and aggregation was promoted by pre-existing seed aggregates and the

4. Conclusions RCL is one of the lipases whose catalytic ability and stability increases after HHP treatment in a moderate range of pressures. CD spectrum confirmed that the secondary structure was highly resistant to pressure, and only began to be lost at pressure over 400 MPa. From atmospheric pressure to 400 MPa, the protein maintained a native-like structure. Above 400 MPa, the tertiary structure was disturbed with a loss of the characteristic signals. A partially unfolded intermediate with a loosely compacted conformation and the exposure of hydrophobic regions were observed at pressures of 600 MPa. It appears that a MG state is reached under this high pressure. In addition, DLS and AFM studies indicated the presence of aggregates formed by a seeding effect. The initial soluble particles played a critical role in the aggregation of RCL. The addition of (NH4)2SO4 inhibited the seeding effect and provided further evidence that in the absence of salt the protein is in a MG state. Acknowledgement This study was financially supported by the Specialized Research Fund for the Doctoral Program of Higher Education (No. 159

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