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Secondary structure and folding stability of proteins adsorbed on silica particles – Pressure versus temperature denaturation Süleyman Cinar, Claus Czeslik ∗ TU Dortmund University, Department of Chemistry and Chemical Biology, D-44221 Dortmund, Germany
a r t i c l e
i n f o
Article history: Received 23 January 2015 Received in revised form 17 March 2015 Accepted 18 March 2015 Available online xxx Keywords: Protein adsorption Protein unfolding FTIR spectroscopy Temperature effects Pressure effects
a b s t r a c t We present a systematic study of the pressure and temperature dependent unfolding behavior of proteins that are adsorbed on silica particles. Hen egg white lysozyme and bovine ribonuclease A (RNase) were used as model proteins, and their secondary structures were resolved by Fourier transform infrared (FTIR) spectroscopy in the temperature range of 10–90 ◦ C and the pressure range of 1–16,000 bar. Apparently, the secondary structures of both proteins do not change significantly when they are adsorbing on the silica particles. Remarkably, the changes of the secondary structure elements upon protein unfolding are very similar in the adsorbed and the free states. This similarity could be observed for both lysozyme and RNase using both high pressures and high temperatures as denaturing conditions. However, the pressures and temperatures of unfolding of lysozyme and RNase are drastically lowered upon adsorption indicating lower folding stabilities of the proteins on the silica particles. Moreover, the temperature ranges, where changes in secondary structure occur, are broadened due to adsorption, which is related to smaller enthalpy changes of unfolding. For both proteins, free or adsorbed, pressure-induced unfolding always leads to less pronounced changes in secondary structure than temperature-induced unfolding. In the case of lysozyme, high pressure also favors a different unfolded conformation than high temperature. Overall, the results of this study reveal that adsorption of proteins on silica particles decreases the folding stability against high pressures and temperatures, whereas the unfolding pathways are mainly preserved in the adsorbed state. © 2015 Elsevier B.V. All rights reserved.
1. Introduction When proteins are used in biotechnological applications, they are often immobilized on carrier particles [1,2]. Using carrier particles allows for an easier way to remove proteins from the reaction solution by centrifugation, sedimentation, filtration or even magnetic forces [3]. For example, expensive enzymes can then be reused in additional reaction cycles. However, proteins are biologically active in their native conformation only, which is marginally stable against unfolding. Any deviation from the natural environment, which is mostly a neutral aqueous solution at ambient pressure and slightly elevated temperatures, can cause denaturation due to partial or complete unfolding. For example, proteins can be unfolded by increasing and lowering the temperature, increasing the pressure, or changing the chemical environment, such as addition of cosolvents or exposure to interfaces [4–7]. Thus, immobilization
of proteins on carrier particles can also lead to a loss of biological activity. From a thermodynamic point of view, the stability of the native, folded conformation is given by the standard Gibbs energy of unfolding, G◦ , which is G◦ unfolded − G◦ folded . It characterizes a two-state model of a folded conformation that is in equilibrium with unfolded conformations via [6] G◦ = −RT ln K
(1)
where K = cunfolded /cfolded is the equilibrium constant. The standard state is given by c◦ = 1 mol L−1 . By increasing the temperature, T, the protein unfolds, when G◦ becomes negative. This temperature dependence can be expressed by [4]
◦
◦
G (T ) = G (Tunf ) +
∂G◦ ∂T
= −S(Tunf ) × (T − Tunf ) ∗ Corresponding author. Tel.: +49 2317553903. E-mail address: [email protected] (C. Czeslik).
× (T − Tunf ) p
(2)
Here, we neglect the second order derivative, which is related to the heat capacity change of unfolding, and we use G◦ (Tunf ) = 0, where Tunf is the temperature of unfolding. S is the entropy change of
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unfolding, which has a positive value. The enthalpy change of unfolding can be derived from H = Tunf S. In a similar way, the effect of pressure, p, on the standard Gibbs energy of unfolding can be written as [4]
◦
◦
G (p) = G (punf ) +
∂G◦ ∂p
× (p − punf ) = V (punf ) T
× (p − punf )
(3) G◦
where punf is the pressure of unfolding and (punf ) = 0. V is the volume change of unfolding, which is usually negative. There are different contributions to V. Most importantly, void volumes (cavities) in the folded conformation of a protein are filled with water molecules upon unfolding, which reduces the total volume of the system, but other contributions are also discussed in literatures [8–10]. Upon adsorption at an aqueous-solid interface, proteins can undergo some changes in conformation and partial unfolding. There are many studies in the literature that characterize these changes in terms of the secondary or the tertiary protein structure [11–20]. Of course, the extent of an adsorption-induced partial unfolding is related to the strength of protein–interface interactions and the folding stability of the protein. However, so far, there seems to be no clear picture or way to predict what kind of secondary structure is formed at aqueous-solid interfaces. On the other hand, there are a few studies in the literature indicating that the temperature range of protein unfolding is broadened and shifted to lower temperatures, when the protein is adsorbed at aqueous-solid interfaces [13,21,22]. Moreover, we have also found recently that the pressure range of protein unfolding is broadened and shifted to lower pressures at interfaces [23]. In this way, adsorption-induced conformational changes of a protein might simply be regarded as the onset of protein unfolding that already occurs at ambient conditions. Therefore, the broad aim of this study is to investigate, if the conformation of an adsorbed protein can be related to any conformation of a free protein under denaturing conditions. In a systematic way, we study two proteins, free in solution and adsorbed on silica particles, and determine their secondary structures in the course of unfolding under conditions of high pressures and high temperatures. Hen egg white lysozyme and ribonuclease A (RNase) are used as model proteins. To unfold a protein by pressure, pressures in the kbar region are needed. Therefore, we have applied the diamond anvil technique. Using Fourier transform infrared (FTIR) spectroscopy, the secondary structures of the proteins under all conditions has been derived from the amide I band, which is composed of subbands arising from the various secondary structure elements of a protein [24]. As we will show here, proteins undergo the same changes in secondary structure in the adsorbed and the free state, when they are unfolded under high pressures or temperatures. The main effect of the interface is only a lowering of the pressure and temperature of unfolding of the proteins. This finding supports the idea that conformations of adsorbed proteins are mostly reflecting the normal unfolding equilibrium that is shifted to the unfolded side. 2. Materials and methods
is overlapping with the bending mode of liquid H2 O. 10 mM phosphate–D2 O buffer with pD = 7.8 (pD = pH-meter reading + 0.4 [25]) and 10 mM Tris–D2 O buffer with pD = 7.8 were used for temperature-dependent and pressure-dependent experiments, respectively. They are characterized by only small pD-shifts when they are heated (phosphate) or pressurized (Tris) [26]. Protein solutions were prepared by dissolving 2 mg of lysozyme or RNase in 200 L of D2 O followed by heating to 68 ◦ C (lysozyme) or 63 ◦ C (RNase) for 15 min. During this heating, labile H atoms of the protein are exchanged for D atoms. After lyophilization, 100 L of D2 O buffer solution (phosphate or Tris) were added to generate a 2 wt% protein solution, which was used to study the free protein in the absence of silica particles. In a similar way, a 10 wt% protein solution was also prepared that was used to adsorb protein molecules on silica particles. Ludox AM colloidal silica (density 1.21 g mL−1 , specific surface area > 198 m2 g−1 ) from Sigma–Aldrich was used as the source for silica particles. The H2 O solvent of this colloidal dispersion has been replaced by D2 O using Amicon Ultra centrifugal filters (500 L, 10 kDa). This type of filter retains the silica particles, and only the solvent can pass. A filter was loaded with a 1:1 mixture of the original colloidal dispersion and D2 O buffer solution. After intense mixing and centrifugation at 10,000 × g for 10 min, the volume was reduced from 500 to 300 L, because 200 L of the solvent had passed through the filter. The loss of solvent was compensated by addition of 200 L of fresh D2 O buffer solution. This procedure was repeated eight times. Finally, no residual H2 O could be detected by FTIR spectroscopy. For protein adsorption on silica particles, the 10 wt% protein solution was mixed with the silica-D2 O dispersion in a volume ratio of 1:4 to generate a 2 wt% protein solution with silica particles that was used in the FTIR experiments. Silica particles have a negative surface charge, whereas the proteins, lysozyme and RNase, have a positive net charge at neutral pD-values (the isoelectric points of lysozyme and RNase are found at pH 11.0 and 9.4, respectively [27,28]). This electrostatic attraction between the protein molecules and the silica particles leads to complete adsorption, as has been confirmed experimentally. After 15 min of incubation, protein-silica sample solutions were filled into Amicon Ultra centrifugal filters (volume 500 L, pore size 50 kDa) and were centrifuged at 10,000 × g for 15 min. In this way, all non-adsorbed protein is passing through the filter, whereas all proteins that are adsorbed on the silica particles are retained by the filter. The filtrate was analyzed by UV-spectroscopy at 270–300 nm, however, no UV-absorption was detected proving that essentially all lysozyme or RNase molecules are adsorbed on the silica particles. The surface coverage of the silica particles can be calculated as follows. Ludox AM colloidal silica has a surface area of at least 239 m2 mL−1 . During sample preparation, the colloidal dispersion has been diluted in two steps by factors of 2 and 1.25 resulting in a surface area of 95 m2 mL−1 . Lysozyme and RNase have molar masses of 14,300 g mol−1 and 13,700 g mol−1 , respectively, and a specific volume of 0.703 mL g−1 [6]. Assuming spherical shapes, we obtain a radius of r = 1.6 × 10−9 m and a molecular footprint area of r2 = 7.8 × 10−18 m2 for both proteins. Samples contain 2 wt% of protein corresponding to 8.6 × 1017 molecules mL−1 . Thus, the surface coverage is 8.6 × 1017 × 7.8 × 10−18 m2 /95 m2 = 0.07 = 7% by lysozyme or RNase.
2.1. Sample preparation 2.2. FTIR spectroscopy Hen egg white lysozyme (product No. 10837059001) was purchased from Roche Diagnostics (Mannheim, Germany) and ribonuclease A from bovine pancreas (RNase; product No. R5500) from Sigma–Aldrich (Steinheim, Germany). All measurements were performed in D2 O buffer solutions, because the amide I band, which is sensitive to the secondary structure of a protein,
Infrared absorption spectra of free and adsorbed lysozyme and RNase were recorded using the Nicolet 6700 Fourier transform infrared spectrometer from Thermo Fisher Scientific operating with a liquid nitrogen-cooled MCT detector. The whole spectrometer was continuously purged with dry air. Temperature-dependent
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Fig. 1. Decomposition of the amide I band of lysozyme in D2 O buffer solution. Each subband represents a secondary structure element. The areas of the subbands are proportional to the fractions of the secondary structure elements.
measurements were performed using a transmission cell with two CaF2 windows that are separated by a 50 m thick Mylar film. The film has a central hole that contains the sample solution (about 20 L). Temperature was controlled using a circulating water flow and was measured directly at the sample. Each FTIR spectrum is the average over 256 interferometer scans with a spectral resolution of 2 cm−1 . Pressure-dependent measurements were performed using the P-series diamond anvil cell from High Pressure Diamond Optics (Tucson, AZ, USA). It has two type IIa diamonds with a surface diameter of 0.6 mm. They are separated by a 50 m thick steel gasket with a central hole of 0.5 mm diameter containing the sample solution (about 10 nL). The temperature of the diamond anvil cell was set to 25 ◦ C by a circulating water flow. Barium sulfate was added to the sample as an internal pressure sensor. The shift of the 983 cm−1 band to higher wavenumbers is proportional to the applied pressure [29]. Background was measured with D2 O buffer solution. Spectral analysis was carried out using the Grams/AI 8.1 software from Thermo Fisher Scientific. After background subtraction, the area of the amide I band between 1700 and 1600 cm−1 was normalized to one. Secondary structure estimation was performed by fitting subbands to the amide I band, whose peak wavenumbers are characteristic for the secondary structure elements and whose areas are proportional their fractions (Fig. 1). Initial peak wavenumbers for the fitting analysis were determined from second derivative spectra and by Fourier self-deconvolution (FSD). Only those peak wavenumbers that emerge from both methods were used for peak fitting. Peak positions were not allowed to move further than 2 cm−1 during peak fitting. The deviations between the measured amide I bands and the fitted curves are very small. 3. Results and discussion 3.1. Secondary structure of lysozyme and RNase as a function of temperature The amide I band (the prime indicates D2 O as the solvent) of proteins in the infrared spectrum between 1700–1600 cm−1 is mainly related to the C O stretching mode of the amide bonds. The exact wavenumber of this mode depends on the type of secondary structure the amide bonds are involved in. Thus, a decomposition of the amide I band into subbands allows for a secondary structure analysis (Fig. 1) [24]. The relative area of a subband is a good estimate for the fraction of the associated secondary structure element.
Fig. 2. Comparison of the normalized amide I bands of (A) lysozyme and (B) RNase, when these proteins are free in solution (black solid lines) and adsorbed on silica particles (red dashed lines) at pD = 7.8, 1 bar, 25 ◦ C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Unfortunately, the amide I band of proteins in H2 O overlaps with the H2 O bending mode. Therefore, all measurements of this study have been carried out with D2 O as the solvent. In Fig. 2, the amide I bands of lysozyme and RNase are shown, when these proteins are free in solution and adsorbed on silica particles. Apparently, the band shape does not change in a significant way upon adsorption of both proteins suggesting similar secondary structures in the free and adsorbed state. Silica particles have a hydrophilic surface, which is favorable for a native protein conformation. Moreover, both proteins are rather stable against unfolding, which is apparent from high temperatures of unfolding ranging at about 70–78 ◦ C for lysozyme [30–32] and about 62–66 ◦ C for RNase [33,34]. Little or no change of the secondary structure upon adsorption on silica is also reported in the literature for lysozyme [35,36] and RNase [11]. Some blue shift of the Trp fluorescence band of lysozyme has been observed upon adsorption on silica particles [22,35]. However, this shift might only indicate a change of the Trp environment due to protein–silica interactions. Indeed, it has also been concluded that lysozyme does not undergo any tertiary structural change upon adsorption on silica [36,37]. Furthermore, lateral protein–protein interactions and aggregation on the silica particles seem to be negligible in our case considering
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secondary structures of free lysozyme (Table 1) and free RNase (Table 2) at 20 ◦ C are in good agreement with the literature. For example, the fraction of ␣-helices in lysozyme was found in this study at 38% (Table 1; 1652 cm−1 ), whereas values of 40–41% are reported [15,17,24]. The -sheets in RNase have a fraction of 50% according to this study (Table 2; sum over subbands at 1633 and 1679 cm−1 ), whereas the same total fraction at these wavenumbers has been observed before [24,38]. Within the experimental error, the same fractions of secondary structure elements are found in the adsorbed state on silica particles at 20 ◦ C (Tables 1 and 2). It is remarkable that even the changes of the secondary structure of free lysozyme upon thermal unfolding are very similar to the changes observed for the unfolding of adsorbed lysozyme (Table 1). For example, the fraction of ␣-helices is reduced by −13% in the case of free lysozyme and by −12% in the case of adsorbed lysozyme. At the same time, the fractions of turns are increasing by +12% and +11%, respectively. In the case of RNase (Table 2), thermal unfolding leads to a loss of −21% of -sheets (1633 cm−1 ), when the protein is free in solution, and −23% in the adsorbed state of the protein. This loss of ordered secondary structure is mostly compensated by the formation of unordered segments, whose fractions increase by +15% and +22%, respectively (Table 2). It is noted that thermally unfolded lysozyme and RNase contain significant amounts of residual ordered secondary structure (Tables 1 and 2), as has been reported for free RNase [38,39]. However, for both proteins, there are similar changes of the secondary structure upon thermal unfolding in the free and the adsorbed state on silica particles. Apparently, protein-silica interactions do not affect the principal unfolding pathway of lysozyme and RNase. To quantify the extent of changes in the secondary structure upon protein unfolding, we define the mean squared change according to 1 2 fi N N
f 2 =
(4)
i=1
Fig. 3. Temperature dependence of the fractional band areas of (A) free lysozyme in solution and (B) adsorbed lysozyme on silica particles. The wavenumbers (in cm−1 ) of the subbands are given in the legends. The measurements were carried out in triplicate. The corresponding data of RNase are given in the Supporting information.
the low surface coverage of the particles of about 7% (see Section 2.1). Some loss of enzymatic activity has been reported for lysozyme upon adsorption on silica particles [15,35]. However, the enzymatic activity of an adsorbed protein is expected to be reduced even if the native structure is preserved, because protein–interface interactions slow down protein dynamics and partially block the active site. By comparing Fig. 2A with Fig. 2B, it becomes visible that the amide I band reflects the composition of the secondary structure elements of a protein. The maximum wavenumber of lysozyme is found at 1649 cm−1 and that of RNase at 1635 cm−1 . Pure ␣-helices and -sheets of proteins are characterized by infrared absorption bands at 1650–1654 cm−1 and 1620–1640 cm−1 , respectively [24]. As shown below, the secondary structure of lysozyme contains mostly ␣-helices, whereas that of RNase is dominated by -sheets. A detailed secondary structure analysis is shown in Fig. 3 for free and adsorbed lysozyme and in Fig. S1 (Supporting information) for free and adsorbed RNase as a function of temperature. The assignment of the resolved subbands to the various secondary structure elements was made according to the literature [24] and is given in Table 1 for lysozyme and Table 2 for RNase. Upon heating and thermal unfolding, both proteins lose much of their ordered secondary structure (i.e. ␣-helices and -sheets), whereas the fractions of turns and unordered segments are increasing. The resolved
where fi is the change (in %) of the fraction of the secondary structure element i as given in Tables 1 and 2. Using Eq. (4), we obtain values of 68 and 51 for free and adsorbed lysozyme, respectively, and values of 134 and 174 for free and adsorbed RNase, respectively. Thus, the thermal unfolding of lysozyme is associated with a much smaller mean squared change of the secondary structure than RNase. This is a reasonable result, because the folded structure of lysozyme is strengthened by four disulfide bridges. 3.2. Secondary structure of lysozyme and RNase as a function of pressure The pressure-induced unfolding of free and adsorbed lysozyme and RNase has also been studied by FTIR spectroscopy. The results of the secondary structure analysis as a function of pressure are given in Figs. 4 and S2 (Supporting information). The amide I bands of lysozyme and RNase have been decomposed in the same way as done in the analysis of the temperature-dependent measurements. The fractions of the secondary structure elements before and after pressure-induced unfolding are also summarized in Tables 1 (lysozyme) and 2 (RNase). In the case of lysozyme, pressure-induced unfolding leads to a loss of −12% and −11% of ␣-helices in the free and adsorbed state of the protein, respectively (Table 1). It is interesting to note that these values are very similar to those found upon thermal unfolding (Table 1). However, in contrast to thermal unfolding, pressure-induced unfolding favors the formation of unordered segments, whose fractions are increasing by +10% (free lysozyme) and +9% (adsorbed lysozyme), instead of turns. This difference of the pressure and temperature effects is already evident from a simple
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Table 1 Fractions of secondary structure elements of lysozyme before and after temperature and pressure induced unfolding in the free and the adsorbed state on silica particles.a 1679 cm−1 b Turnc
1666 cm−1 b Turn
1652 cm−1 b ␣-Helix
1640 cm−1 b Unordered
1630 cm−1 b -Sheet
1613 cm−1 b Baselined
Free, 20 ◦ C Free, 90 ◦ C Change
6 12 +6
13 25 +12
38 25 −13
20 23 +3
20 13 −7
3 2 −1
Ads., 20 ◦ C Ads., 90 ◦ C Change
8 8 0
13 24 +11
36 24 −12
19 22 +3
20 15 −5
4 7 +3
Free, 5 kbar Free, 12 kbar Change
8 8 0
12 16 +4
37 25 −12
18 28 +10
18 16 −2
7 7 0
Ads., 1 kbar Ads., 8 kbar Change
9 9 0
13 15 +2
35 24 −11
19 28 +9
17 17 0
7 7 0
a b c d
Fractions are given in percent and were found to vary by ±2% over three measurements. Maximum changes are printed in bold. Wavenumbers are starting values for band fitting and were allowed to move by ±2 cm−1 . Subband also indicates -sheets. Subband used for minor baseline correction.
comparison of the amide I band shapes of lysozyme under high pressures and high temperatures (Fig. S3, Supporting information). An increase of the temperature (Fig. S3A) leads to an increased infrared absorption at higher wavenumbers, whereas an increase of the pressure (Fig. S3B) induces increased infrared absorption at lower wavenumbers. The principal origin of different pressure and temperature effects on protein unfolding can be found in Eqs. (2) and (3). Proteins unfold under high temperatures, because the unfolded state has a higher entropy than the folded state, whereas low-volume conformations of a protein are selected under high pressures. Apparently, in the case of lysozyme unfolding, the formation of turns can raise the entropy but cannot lower the volume sufficiently. However, although pressure seems to guide lysozyme on a different unfolding pathway than temperature, it becomes apparent that the pressure-induced unfolding of lysozyme follows the same route in the free and the adsorbed state. Therefore, as found in the temperature-dependent measurements, protein–silica interactions do not affect the principal pressuredependent unfolding pathway of lysozyme. Finally, we also find that pressure-induced unfolding of RNase is also very similar in the free and the adsorbed state of the protein in terms of secondary structural changes (Table 2).
There are decreasing -sheet fractions (subband at 1633 cm−1 ; −10% for free RNase, −11% for adsorbed RNase), which are roughly compensated by increasing fractions of unordered segments (subband at 1644 cm−1 ; +15% for free RNase, +16% for adsorbed RNase). The fractions of all other secondary structure elements also show almost identical changes in the free and the adsorbed state of RNase upon pressure-induced unfolding (Table 2). Thus, as found for lysozyme, the unfolding pathway of RNase under high pressure appears to be independent of protein–silica interactions. Eq. (4) can also be used to point out a general difference between temperature and pressure effects on protein unfolding, which has already been described in other studies [38,40]. High pressure represents a mild denaturing condition causing less structural changes of proteins than high temperature. For example, the mean squared change (Eq. (4)) of secondary structure of free RNase is 134 at high T and only 67 at high p, that of adsorbed RNase is 174 at high T and only 69 at high p. Thus, pressure-induced unfolding of RNase involves much less structural change than temperature-induced unfolding. Moreover, the results of this study show that this general observation can be confirmed for proteins that are adsorbed at the aqueous–silica interface.
Table 2 Fractions of secondary structure elements of RNase before and after temperature and pressure induced unfolding in the free and the adsorbed state on silica particles.a 1679 cm−1 b -Sheetc
1670 cm−1 b Turn
1663 cm−1 b Turn
1653 cm−1 b ␣-Helix
1644 cm−1 b Unordered
1633 cm−1 b -Sheet
1613 cm−1 b Baselined
Free, 20 ◦ C Free, 80 ◦ C Change
9 7 −2
6 14 +8
8 19 +11
21 12 −9
11 26 +15
41 20 −21
4 2 −2
Ads., 20 ◦ C Ads., 80 ◦ C Change
11 9 −2
6 13 +7
8 16 +8
20 11 −9
8 30 +22
42 19 −23
5 2 −3
Free, 5 kbar Free, 12 kbar Change
10 4 −6
6 10 +4
8 13 +5
21 13 −8
10 25 +15
42 32 −10
3 3 0
Ads., 1 kbar Ads., 8 kbar Change
11 6 −5
6 10 +4
9 13 +4
20 13 −7
8 24 +16
42 31 −11
4 3 −1
a b c d
Fractions are given in percent and were found to vary by ±2% over three measurements. Maximum changes are printed in bold. Wavenumbers are starting values for band fitting and were allowed to move by ±2 cm−1 . Subband also indicates turns. Subband used for minor baseline correction.
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Fig. 4. Pressure dependence of the fractional band areas of (A) free lysozyme in solution and (B) adsorbed lysozyme on silica particles. The wavenumbers (in cm−1 ) of the subbands are given in the legends. The measurements were performed in triplicate. The corresponding data of RNase are given in the Supporting information.
3.3. Folding stability of lysozyme and RNase adsorbed on silica particles Based on the results of Sections 3.1 and 3.2, the adsorption on silica particles has little effect on the unfolding pathway of lysozyme and RNase, as probed by increased temperatures and pressures. However, Figs. 3, 4, S1 and S2 clearly show that the temperature of unfolding, Tunf , and the pressure of unfolding, punf , are reduced drastically, when lysozyme and RNase adsorb on silica particles. For a precise determination of Tunf and punf , we have used the following equations (which can be derived from Eqs. (1)–(3)): A=
AF − AU + AU 1 + exp[−(H/R)(1/T − 1/Tunf )]
(5)
A=
AF − AU + AU 1 + exp[−(V/RT )(p − punf )]
(6)
where A is the infrared absorbance at a selected wavenumber. We have chosen 1644 cm−1 in the case of lysozyme and 1633 cm−1 in the case of RNase, because there is a relatively large change of A during unfolding at these wavenumbers. AF and AU are the plateau values for the folded and the unfolded state. A was plotted as a function of inverse temperature, 1/T, or pressure, p, and fitted with
Eq. (5) or (6), respectively, for free and adsorbed lysozyme and RNase (Fig. 5). It is important to note that the use of Eqs. (5) and (6) assumes equilibrium conditions and reversible transitions. Unfolding transitions of free lysozyme and free RNase were found to be reversible as a function of temperature and pressure, as judged from the amide I band shapes. In the adsorbed state, full refolding was found on the pressure axis, whereas incomplete refolding was found on the temperature axis, which may indicate very slow refolding kinetics due to multiple protein–silica contacts. However, during heating or pressurization scans, the infrared absorbance, A, was recorded for the preset temperature or pressure, after A had reached a constant value, which usually took 10–20 min. As can be seen from Fig. 5A and B, the temperature of unfolding (fraction folded = 0.5) of lysozyme is reduced from 78 to 63 ◦ C due to adsorption on silica particles, whereas that of RNase is reduced from 66 to 56 ◦ C. The pressures of unfolding are also reduced (fraction folded = 0.5 in Fig. 5C and D). In the case of lysozyme, adsorption leads to a reduction from 9.0 to 5.1 kbar, and in the case of RNase, we observe a drop from 7.7 to 4.7 kbar. Thus, the folded conformations of lysozyme and RNase are less stable against increased temperature and pressure in the adsorbed states of the proteins. For comparison, pressures of unfolding of 9.6 and 7.4 kbar are reported for free lysozyme [41] and free RNase [38], respectively, in good agreement with our values. Furthermore, from fitting Eqs. (5) and (6) to the measured infrared absorbance, A, the enthalpy change of unfolding, H, and the volume change of unfolding, V, are also obtained. These two thermodynamic parameters are proportional to the slopes at the inflection points in Fig. 5. Upon adsorption on silica particles, H of lysozyme is decreasing from 385 to 244 kJ mol−1 , and H of RNase is decreasing from 368 to 209 kJ mol−1 . This reduction can simply be explained by protein–silica interactions. H is the difference HU − HF , where the subscripts indicate the unfolded and the folded state of the protein. A stronger attractive protein–silica interaction of the unfolded state, as compared to the folded state, will reduce HU in a stronger way than HF , resulting in a decreased H value. The smaller H values observed in the adsorbed state of lysozyme and RNase are responsible for the broader temperature ranges of unfolding (Fig. 5A and B). Unfortunately, the V values, which can be derived from the fits in Fig. 5C and D, have a large error (about ±50%), because the precision of the pressure determination of the diamond anvil technique used here is rather low (about ±200 bar). Anyway, we find values of −21 mL mol−1 (free lysozyme), −24 mL mol−1 (adsorbed lysozyme), −123 mL mol−1 (free RNase), and −36 mL mol−1 (adsorbed RNase). In the literature, for example, values of −26 mL mol−1 [41] and −55 mL mol−1 [42] are reported for free lysozyme, and values of −45 mL mol−1 [40] and −15 mL mol−1 [34] are reported for free RNase illustrating the experimental difficulty to obtain precise V values. Recently, we have studied the pressure-induced unfolding of staphylococcal nuclease (SNase) by fluorescence spectroscopy. The volume change of unfolding, V, was found to be −73 mL mol−1 in the free state and −41 mL mol−1 , when SNase is adsorbed on silica particles [23]. This reduction of |V| (absolute value because of negative sign) upon protein adsorption has been explained by a negative volume change of adsorption [23]. A small negative volume change of adsorption has also been found for lysozyme at the silica–water interface using fluorescence spectroscopy and neutron reflectometry [43], suggesting an absolute volume change of unfolding, |V|, that is smaller for adsorbed lysozyme. Despite all these experimental challenges, there seems to be a trend that both H and |V| are smaller in the adsorbed state of a protein. And, smaller H and |V| values in the adsorbed state are equivalent to broader temperature and pressure ranges, where the protein unfolds.
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Fig. 5. Adsorption-induced destabilization of the folded conformation of lysozyme and RNase as probed by heating and pressurization. The absorbance of lysozyme at 1644 cm−1 and that of RNase at 1633 cm−1 has been measured and rescaled to cover the range from 0 to 1.
4. Conclusions
References
All data obtained in this study point to a rather simple mechanism of adsorption-induced conformational changes of proteins. Whereas the unfolding pathway of a protein on the temperature or pressure axis remains largely unaltered upon adsorption, the folding stability is significantly reduced in the adsorbed state leading to broader unfolding ranges that are shifted to lower temperatures and pressures. Although we could observe this behavior for two proteins and two denaturing conditions, the adsorbent material is limited in this study to silica particles, which are characterized by a negative surface charge and a relatively high hydrophilicity. In this way, protein–interface interactions are dominated by electrostatic forces, and the hydration of the adsorbed protein is always similar to that of the free protein. These circumstances are probably favorable to preserve the conformational space of a protein. On the other hand, when proteins are adsorbed on hydrophobic surfaces, they may undergo a different unfolding pathway as compared to the solution, because conformational substates, which are highly unfavorable in aqueous solution, could be stabilized by hydrophobic interactions between the protein and the interface.
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Acknowledgment This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) (grant number Cz 77/3-1). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2015.03.043.
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Secondary structure and folding stability of proteins adsorbed on silica particles – Pressure versus temperature denaturation Süleyman Cinar, Claus Czeslik ∗ TU Dortmund University, Department of Chemistry and Chemical Biology, D-44221 Dortmund, Germany
a r t i c l e
i n f o
Article history: Received 23 January 2015 Received in revised form 17 March 2015 Accepted 18 March 2015 Available online xxx Keywords: Protein adsorption Protein unfolding FTIR spectroscopy Temperature effects Pressure effects
a b s t r a c t We present a systematic study of the pressure and temperature dependent unfolding behavior of proteins that are adsorbed on silica particles. Hen egg white lysozyme and bovine ribonuclease A (RNase) were used as model proteins, and their secondary structures were resolved by Fourier transform infrared (FTIR) spectroscopy in the temperature range of 10–90 ◦ C and the pressure range of 1–16,000 bar. Apparently, the secondary structures of both proteins do not change significantly when they are adsorbing on the silica particles. Remarkably, the changes of the secondary structure elements upon protein unfolding are very similar in the adsorbed and the free states. This similarity could be observed for both lysozyme and RNase using both high pressures and high temperatures as denaturing conditions. However, the pressures and temperatures of unfolding of lysozyme and RNase are drastically lowered upon adsorption indicating lower folding stabilities of the proteins on the silica particles. Moreover, the temperature ranges, where changes in secondary structure occur, are broadened due to adsorption, which is related to smaller enthalpy changes of unfolding. For both proteins, free or adsorbed, pressure-induced unfolding always leads to less pronounced changes in secondary structure than temperature-induced unfolding. In the case of lysozyme, high pressure also favors a different unfolded conformation than high temperature. Overall, the results of this study reveal that adsorption of proteins on silica particles decreases the folding stability against high pressures and temperatures, whereas the unfolding pathways are mainly preserved in the adsorbed state. © 2015 Elsevier B.V. All rights reserved.
1. Introduction When proteins are used in biotechnological applications, they are often immobilized on carrier particles [1,2]. Using carrier particles allows for an easier way to remove proteins from the reaction solution by centrifugation, sedimentation, filtration or even magnetic forces [3]. For example, expensive enzymes can then be reused in additional reaction cycles. However, proteins are biologically active in their native conformation only, which is marginally stable against unfolding. Any deviation from the natural environment, which is mostly a neutral aqueous solution at ambient pressure and slightly elevated temperatures, can cause denaturation due to partial or complete unfolding. For example, proteins can be unfolded by increasing and lowering the temperature, increasing the pressure, or changing the chemical environment, such as addition of cosolvents or exposure to interfaces [4–7]. Thus, immobilization
of proteins on carrier particles can also lead to a loss of biological activity. From a thermodynamic point of view, the stability of the native, folded conformation is given by the standard Gibbs energy of unfolding, G◦ , which is G◦ unfolded − G◦ folded . It characterizes a two-state model of a folded conformation that is in equilibrium with unfolded conformations via [6] G◦ = −RT ln K
(1)
where K = cunfolded /cfolded is the equilibrium constant. The standard state is given by c◦ = 1 mol L−1 . By increasing the temperature, T, the protein unfolds, when G◦ becomes negative. This temperature dependence can be expressed by [4]
◦
◦
G (T ) = G (Tunf ) +
∂G◦ ∂T
= −S(Tunf ) × (T − Tunf ) ∗ Corresponding author. Tel.: +49 2317553903. E-mail address: [email protected] (C. Czeslik).
× (T − Tunf ) p
(2)
Here, we neglect the second order derivative, which is related to the heat capacity change of unfolding, and we use G◦ (Tunf ) = 0, where Tunf is the temperature of unfolding. S is the entropy change of
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unfolding, which has a positive value. The enthalpy change of unfolding can be derived from H = Tunf S. In a similar way, the effect of pressure, p, on the standard Gibbs energy of unfolding can be written as [4]
◦
◦
G (p) = G (punf ) +
∂G◦ ∂p
× (p − punf ) = V (punf ) T
× (p − punf )
(3) G◦
where punf is the pressure of unfolding and (punf ) = 0. V is the volume change of unfolding, which is usually negative. There are different contributions to V. Most importantly, void volumes (cavities) in the folded conformation of a protein are filled with water molecules upon unfolding, which reduces the total volume of the system, but other contributions are also discussed in literatures [8–10]. Upon adsorption at an aqueous-solid interface, proteins can undergo some changes in conformation and partial unfolding. There are many studies in the literature that characterize these changes in terms of the secondary or the tertiary protein structure [11–20]. Of course, the extent of an adsorption-induced partial unfolding is related to the strength of protein–interface interactions and the folding stability of the protein. However, so far, there seems to be no clear picture or way to predict what kind of secondary structure is formed at aqueous-solid interfaces. On the other hand, there are a few studies in the literature indicating that the temperature range of protein unfolding is broadened and shifted to lower temperatures, when the protein is adsorbed at aqueous-solid interfaces [13,21,22]. Moreover, we have also found recently that the pressure range of protein unfolding is broadened and shifted to lower pressures at interfaces [23]. In this way, adsorption-induced conformational changes of a protein might simply be regarded as the onset of protein unfolding that already occurs at ambient conditions. Therefore, the broad aim of this study is to investigate, if the conformation of an adsorbed protein can be related to any conformation of a free protein under denaturing conditions. In a systematic way, we study two proteins, free in solution and adsorbed on silica particles, and determine their secondary structures in the course of unfolding under conditions of high pressures and high temperatures. Hen egg white lysozyme and ribonuclease A (RNase) are used as model proteins. To unfold a protein by pressure, pressures in the kbar region are needed. Therefore, we have applied the diamond anvil technique. Using Fourier transform infrared (FTIR) spectroscopy, the secondary structures of the proteins under all conditions has been derived from the amide I band, which is composed of subbands arising from the various secondary structure elements of a protein [24]. As we will show here, proteins undergo the same changes in secondary structure in the adsorbed and the free state, when they are unfolded under high pressures or temperatures. The main effect of the interface is only a lowering of the pressure and temperature of unfolding of the proteins. This finding supports the idea that conformations of adsorbed proteins are mostly reflecting the normal unfolding equilibrium that is shifted to the unfolded side. 2. Materials and methods
is overlapping with the bending mode of liquid H2 O. 10 mM phosphate–D2 O buffer with pD = 7.8 (pD = pH-meter reading + 0.4 [25]) and 10 mM Tris–D2 O buffer with pD = 7.8 were used for temperature-dependent and pressure-dependent experiments, respectively. They are characterized by only small pD-shifts when they are heated (phosphate) or pressurized (Tris) [26]. Protein solutions were prepared by dissolving 2 mg of lysozyme or RNase in 200 L of D2 O followed by heating to 68 ◦ C (lysozyme) or 63 ◦ C (RNase) for 15 min. During this heating, labile H atoms of the protein are exchanged for D atoms. After lyophilization, 100 L of D2 O buffer solution (phosphate or Tris) were added to generate a 2 wt% protein solution, which was used to study the free protein in the absence of silica particles. In a similar way, a 10 wt% protein solution was also prepared that was used to adsorb protein molecules on silica particles. Ludox AM colloidal silica (density 1.21 g mL−1 , specific surface area > 198 m2 g−1 ) from Sigma–Aldrich was used as the source for silica particles. The H2 O solvent of this colloidal dispersion has been replaced by D2 O using Amicon Ultra centrifugal filters (500 L, 10 kDa). This type of filter retains the silica particles, and only the solvent can pass. A filter was loaded with a 1:1 mixture of the original colloidal dispersion and D2 O buffer solution. After intense mixing and centrifugation at 10,000 × g for 10 min, the volume was reduced from 500 to 300 L, because 200 L of the solvent had passed through the filter. The loss of solvent was compensated by addition of 200 L of fresh D2 O buffer solution. This procedure was repeated eight times. Finally, no residual H2 O could be detected by FTIR spectroscopy. For protein adsorption on silica particles, the 10 wt% protein solution was mixed with the silica-D2 O dispersion in a volume ratio of 1:4 to generate a 2 wt% protein solution with silica particles that was used in the FTIR experiments. Silica particles have a negative surface charge, whereas the proteins, lysozyme and RNase, have a positive net charge at neutral pD-values (the isoelectric points of lysozyme and RNase are found at pH 11.0 and 9.4, respectively [27,28]). This electrostatic attraction between the protein molecules and the silica particles leads to complete adsorption, as has been confirmed experimentally. After 15 min of incubation, protein-silica sample solutions were filled into Amicon Ultra centrifugal filters (volume 500 L, pore size 50 kDa) and were centrifuged at 10,000 × g for 15 min. In this way, all non-adsorbed protein is passing through the filter, whereas all proteins that are adsorbed on the silica particles are retained by the filter. The filtrate was analyzed by UV-spectroscopy at 270–300 nm, however, no UV-absorption was detected proving that essentially all lysozyme or RNase molecules are adsorbed on the silica particles. The surface coverage of the silica particles can be calculated as follows. Ludox AM colloidal silica has a surface area of at least 239 m2 mL−1 . During sample preparation, the colloidal dispersion has been diluted in two steps by factors of 2 and 1.25 resulting in a surface area of 95 m2 mL−1 . Lysozyme and RNase have molar masses of 14,300 g mol−1 and 13,700 g mol−1 , respectively, and a specific volume of 0.703 mL g−1 [6]. Assuming spherical shapes, we obtain a radius of r = 1.6 × 10−9 m and a molecular footprint area of r2 = 7.8 × 10−18 m2 for both proteins. Samples contain 2 wt% of protein corresponding to 8.6 × 1017 molecules mL−1 . Thus, the surface coverage is 8.6 × 1017 × 7.8 × 10−18 m2 /95 m2 = 0.07 = 7% by lysozyme or RNase.
2.1. Sample preparation 2.2. FTIR spectroscopy Hen egg white lysozyme (product No. 10837059001) was purchased from Roche Diagnostics (Mannheim, Germany) and ribonuclease A from bovine pancreas (RNase; product No. R5500) from Sigma–Aldrich (Steinheim, Germany). All measurements were performed in D2 O buffer solutions, because the amide I band, which is sensitive to the secondary structure of a protein,
Infrared absorption spectra of free and adsorbed lysozyme and RNase were recorded using the Nicolet 6700 Fourier transform infrared spectrometer from Thermo Fisher Scientific operating with a liquid nitrogen-cooled MCT detector. The whole spectrometer was continuously purged with dry air. Temperature-dependent
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Fig. 1. Decomposition of the amide I band of lysozyme in D2 O buffer solution. Each subband represents a secondary structure element. The areas of the subbands are proportional to the fractions of the secondary structure elements.
measurements were performed using a transmission cell with two CaF2 windows that are separated by a 50 m thick Mylar film. The film has a central hole that contains the sample solution (about 20 L). Temperature was controlled using a circulating water flow and was measured directly at the sample. Each FTIR spectrum is the average over 256 interferometer scans with a spectral resolution of 2 cm−1 . Pressure-dependent measurements were performed using the P-series diamond anvil cell from High Pressure Diamond Optics (Tucson, AZ, USA). It has two type IIa diamonds with a surface diameter of 0.6 mm. They are separated by a 50 m thick steel gasket with a central hole of 0.5 mm diameter containing the sample solution (about 10 nL). The temperature of the diamond anvil cell was set to 25 ◦ C by a circulating water flow. Barium sulfate was added to the sample as an internal pressure sensor. The shift of the 983 cm−1 band to higher wavenumbers is proportional to the applied pressure [29]. Background was measured with D2 O buffer solution. Spectral analysis was carried out using the Grams/AI 8.1 software from Thermo Fisher Scientific. After background subtraction, the area of the amide I band between 1700 and 1600 cm−1 was normalized to one. Secondary structure estimation was performed by fitting subbands to the amide I band, whose peak wavenumbers are characteristic for the secondary structure elements and whose areas are proportional their fractions (Fig. 1). Initial peak wavenumbers for the fitting analysis were determined from second derivative spectra and by Fourier self-deconvolution (FSD). Only those peak wavenumbers that emerge from both methods were used for peak fitting. Peak positions were not allowed to move further than 2 cm−1 during peak fitting. The deviations between the measured amide I bands and the fitted curves are very small. 3. Results and discussion 3.1. Secondary structure of lysozyme and RNase as a function of temperature The amide I band (the prime indicates D2 O as the solvent) of proteins in the infrared spectrum between 1700–1600 cm−1 is mainly related to the C O stretching mode of the amide bonds. The exact wavenumber of this mode depends on the type of secondary structure the amide bonds are involved in. Thus, a decomposition of the amide I band into subbands allows for a secondary structure analysis (Fig. 1) [24]. The relative area of a subband is a good estimate for the fraction of the associated secondary structure element.
Fig. 2. Comparison of the normalized amide I bands of (A) lysozyme and (B) RNase, when these proteins are free in solution (black solid lines) and adsorbed on silica particles (red dashed lines) at pD = 7.8, 1 bar, 25 ◦ C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Unfortunately, the amide I band of proteins in H2 O overlaps with the H2 O bending mode. Therefore, all measurements of this study have been carried out with D2 O as the solvent. In Fig. 2, the amide I bands of lysozyme and RNase are shown, when these proteins are free in solution and adsorbed on silica particles. Apparently, the band shape does not change in a significant way upon adsorption of both proteins suggesting similar secondary structures in the free and adsorbed state. Silica particles have a hydrophilic surface, which is favorable for a native protein conformation. Moreover, both proteins are rather stable against unfolding, which is apparent from high temperatures of unfolding ranging at about 70–78 ◦ C for lysozyme [30–32] and about 62–66 ◦ C for RNase [33,34]. Little or no change of the secondary structure upon adsorption on silica is also reported in the literature for lysozyme [35,36] and RNase [11]. Some blue shift of the Trp fluorescence band of lysozyme has been observed upon adsorption on silica particles [22,35]. However, this shift might only indicate a change of the Trp environment due to protein–silica interactions. Indeed, it has also been concluded that lysozyme does not undergo any tertiary structural change upon adsorption on silica [36,37]. Furthermore, lateral protein–protein interactions and aggregation on the silica particles seem to be negligible in our case considering
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secondary structures of free lysozyme (Table 1) and free RNase (Table 2) at 20 ◦ C are in good agreement with the literature. For example, the fraction of ␣-helices in lysozyme was found in this study at 38% (Table 1; 1652 cm−1 ), whereas values of 40–41% are reported [15,17,24]. The -sheets in RNase have a fraction of 50% according to this study (Table 2; sum over subbands at 1633 and 1679 cm−1 ), whereas the same total fraction at these wavenumbers has been observed before [24,38]. Within the experimental error, the same fractions of secondary structure elements are found in the adsorbed state on silica particles at 20 ◦ C (Tables 1 and 2). It is remarkable that even the changes of the secondary structure of free lysozyme upon thermal unfolding are very similar to the changes observed for the unfolding of adsorbed lysozyme (Table 1). For example, the fraction of ␣-helices is reduced by −13% in the case of free lysozyme and by −12% in the case of adsorbed lysozyme. At the same time, the fractions of turns are increasing by +12% and +11%, respectively. In the case of RNase (Table 2), thermal unfolding leads to a loss of −21% of -sheets (1633 cm−1 ), when the protein is free in solution, and −23% in the adsorbed state of the protein. This loss of ordered secondary structure is mostly compensated by the formation of unordered segments, whose fractions increase by +15% and +22%, respectively (Table 2). It is noted that thermally unfolded lysozyme and RNase contain significant amounts of residual ordered secondary structure (Tables 1 and 2), as has been reported for free RNase [38,39]. However, for both proteins, there are similar changes of the secondary structure upon thermal unfolding in the free and the adsorbed state on silica particles. Apparently, protein-silica interactions do not affect the principal unfolding pathway of lysozyme and RNase. To quantify the extent of changes in the secondary structure upon protein unfolding, we define the mean squared change according to 1 2 fi N N
f 2 =
(4)
i=1
Fig. 3. Temperature dependence of the fractional band areas of (A) free lysozyme in solution and (B) adsorbed lysozyme on silica particles. The wavenumbers (in cm−1 ) of the subbands are given in the legends. The measurements were carried out in triplicate. The corresponding data of RNase are given in the Supporting information.
the low surface coverage of the particles of about 7% (see Section 2.1). Some loss of enzymatic activity has been reported for lysozyme upon adsorption on silica particles [15,35]. However, the enzymatic activity of an adsorbed protein is expected to be reduced even if the native structure is preserved, because protein–interface interactions slow down protein dynamics and partially block the active site. By comparing Fig. 2A with Fig. 2B, it becomes visible that the amide I band reflects the composition of the secondary structure elements of a protein. The maximum wavenumber of lysozyme is found at 1649 cm−1 and that of RNase at 1635 cm−1 . Pure ␣-helices and -sheets of proteins are characterized by infrared absorption bands at 1650–1654 cm−1 and 1620–1640 cm−1 , respectively [24]. As shown below, the secondary structure of lysozyme contains mostly ␣-helices, whereas that of RNase is dominated by -sheets. A detailed secondary structure analysis is shown in Fig. 3 for free and adsorbed lysozyme and in Fig. S1 (Supporting information) for free and adsorbed RNase as a function of temperature. The assignment of the resolved subbands to the various secondary structure elements was made according to the literature [24] and is given in Table 1 for lysozyme and Table 2 for RNase. Upon heating and thermal unfolding, both proteins lose much of their ordered secondary structure (i.e. ␣-helices and -sheets), whereas the fractions of turns and unordered segments are increasing. The resolved
where fi is the change (in %) of the fraction of the secondary structure element i as given in Tables 1 and 2. Using Eq. (4), we obtain values of 68 and 51 for free and adsorbed lysozyme, respectively, and values of 134 and 174 for free and adsorbed RNase, respectively. Thus, the thermal unfolding of lysozyme is associated with a much smaller mean squared change of the secondary structure than RNase. This is a reasonable result, because the folded structure of lysozyme is strengthened by four disulfide bridges. 3.2. Secondary structure of lysozyme and RNase as a function of pressure The pressure-induced unfolding of free and adsorbed lysozyme and RNase has also been studied by FTIR spectroscopy. The results of the secondary structure analysis as a function of pressure are given in Figs. 4 and S2 (Supporting information). The amide I bands of lysozyme and RNase have been decomposed in the same way as done in the analysis of the temperature-dependent measurements. The fractions of the secondary structure elements before and after pressure-induced unfolding are also summarized in Tables 1 (lysozyme) and 2 (RNase). In the case of lysozyme, pressure-induced unfolding leads to a loss of −12% and −11% of ␣-helices in the free and adsorbed state of the protein, respectively (Table 1). It is interesting to note that these values are very similar to those found upon thermal unfolding (Table 1). However, in contrast to thermal unfolding, pressure-induced unfolding favors the formation of unordered segments, whose fractions are increasing by +10% (free lysozyme) and +9% (adsorbed lysozyme), instead of turns. This difference of the pressure and temperature effects is already evident from a simple
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Table 1 Fractions of secondary structure elements of lysozyme before and after temperature and pressure induced unfolding in the free and the adsorbed state on silica particles.a 1679 cm−1 b Turnc
1666 cm−1 b Turn
1652 cm−1 b ␣-Helix
1640 cm−1 b Unordered
1630 cm−1 b -Sheet
1613 cm−1 b Baselined
Free, 20 ◦ C Free, 90 ◦ C Change
6 12 +6
13 25 +12
38 25 −13
20 23 +3
20 13 −7
3 2 −1
Ads., 20 ◦ C Ads., 90 ◦ C Change
8 8 0
13 24 +11
36 24 −12
19 22 +3
20 15 −5
4 7 +3
Free, 5 kbar Free, 12 kbar Change
8 8 0
12 16 +4
37 25 −12
18 28 +10
18 16 −2
7 7 0
Ads., 1 kbar Ads., 8 kbar Change
9 9 0
13 15 +2
35 24 −11
19 28 +9
17 17 0
7 7 0
a b c d
Fractions are given in percent and were found to vary by ±2% over three measurements. Maximum changes are printed in bold. Wavenumbers are starting values for band fitting and were allowed to move by ±2 cm−1 . Subband also indicates -sheets. Subband used for minor baseline correction.
comparison of the amide I band shapes of lysozyme under high pressures and high temperatures (Fig. S3, Supporting information). An increase of the temperature (Fig. S3A) leads to an increased infrared absorption at higher wavenumbers, whereas an increase of the pressure (Fig. S3B) induces increased infrared absorption at lower wavenumbers. The principal origin of different pressure and temperature effects on protein unfolding can be found in Eqs. (2) and (3). Proteins unfold under high temperatures, because the unfolded state has a higher entropy than the folded state, whereas low-volume conformations of a protein are selected under high pressures. Apparently, in the case of lysozyme unfolding, the formation of turns can raise the entropy but cannot lower the volume sufficiently. However, although pressure seems to guide lysozyme on a different unfolding pathway than temperature, it becomes apparent that the pressure-induced unfolding of lysozyme follows the same route in the free and the adsorbed state. Therefore, as found in the temperature-dependent measurements, protein–silica interactions do not affect the principal pressuredependent unfolding pathway of lysozyme. Finally, we also find that pressure-induced unfolding of RNase is also very similar in the free and the adsorbed state of the protein in terms of secondary structural changes (Table 2).
There are decreasing -sheet fractions (subband at 1633 cm−1 ; −10% for free RNase, −11% for adsorbed RNase), which are roughly compensated by increasing fractions of unordered segments (subband at 1644 cm−1 ; +15% for free RNase, +16% for adsorbed RNase). The fractions of all other secondary structure elements also show almost identical changes in the free and the adsorbed state of RNase upon pressure-induced unfolding (Table 2). Thus, as found for lysozyme, the unfolding pathway of RNase under high pressure appears to be independent of protein–silica interactions. Eq. (4) can also be used to point out a general difference between temperature and pressure effects on protein unfolding, which has already been described in other studies [38,40]. High pressure represents a mild denaturing condition causing less structural changes of proteins than high temperature. For example, the mean squared change (Eq. (4)) of secondary structure of free RNase is 134 at high T and only 67 at high p, that of adsorbed RNase is 174 at high T and only 69 at high p. Thus, pressure-induced unfolding of RNase involves much less structural change than temperature-induced unfolding. Moreover, the results of this study show that this general observation can be confirmed for proteins that are adsorbed at the aqueous–silica interface.
Table 2 Fractions of secondary structure elements of RNase before and after temperature and pressure induced unfolding in the free and the adsorbed state on silica particles.a 1679 cm−1 b -Sheetc
1670 cm−1 b Turn
1663 cm−1 b Turn
1653 cm−1 b ␣-Helix
1644 cm−1 b Unordered
1633 cm−1 b -Sheet
1613 cm−1 b Baselined
Free, 20 ◦ C Free, 80 ◦ C Change
9 7 −2
6 14 +8
8 19 +11
21 12 −9
11 26 +15
41 20 −21
4 2 −2
Ads., 20 ◦ C Ads., 80 ◦ C Change
11 9 −2
6 13 +7
8 16 +8
20 11 −9
8 30 +22
42 19 −23
5 2 −3
Free, 5 kbar Free, 12 kbar Change
10 4 −6
6 10 +4
8 13 +5
21 13 −8
10 25 +15
42 32 −10
3 3 0
Ads., 1 kbar Ads., 8 kbar Change
11 6 −5
6 10 +4
9 13 +4
20 13 −7
8 24 +16
42 31 −11
4 3 −1
a b c d
Fractions are given in percent and were found to vary by ±2% over three measurements. Maximum changes are printed in bold. Wavenumbers are starting values for band fitting and were allowed to move by ±2 cm−1 . Subband also indicates turns. Subband used for minor baseline correction.
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Fig. 4. Pressure dependence of the fractional band areas of (A) free lysozyme in solution and (B) adsorbed lysozyme on silica particles. The wavenumbers (in cm−1 ) of the subbands are given in the legends. The measurements were performed in triplicate. The corresponding data of RNase are given in the Supporting information.
3.3. Folding stability of lysozyme and RNase adsorbed on silica particles Based on the results of Sections 3.1 and 3.2, the adsorption on silica particles has little effect on the unfolding pathway of lysozyme and RNase, as probed by increased temperatures and pressures. However, Figs. 3, 4, S1 and S2 clearly show that the temperature of unfolding, Tunf , and the pressure of unfolding, punf , are reduced drastically, when lysozyme and RNase adsorb on silica particles. For a precise determination of Tunf and punf , we have used the following equations (which can be derived from Eqs. (1)–(3)): A=
AF − AU + AU 1 + exp[−(H/R)(1/T − 1/Tunf )]
(5)
A=
AF − AU + AU 1 + exp[−(V/RT )(p − punf )]
(6)
where A is the infrared absorbance at a selected wavenumber. We have chosen 1644 cm−1 in the case of lysozyme and 1633 cm−1 in the case of RNase, because there is a relatively large change of A during unfolding at these wavenumbers. AF and AU are the plateau values for the folded and the unfolded state. A was plotted as a function of inverse temperature, 1/T, or pressure, p, and fitted with
Eq. (5) or (6), respectively, for free and adsorbed lysozyme and RNase (Fig. 5). It is important to note that the use of Eqs. (5) and (6) assumes equilibrium conditions and reversible transitions. Unfolding transitions of free lysozyme and free RNase were found to be reversible as a function of temperature and pressure, as judged from the amide I band shapes. In the adsorbed state, full refolding was found on the pressure axis, whereas incomplete refolding was found on the temperature axis, which may indicate very slow refolding kinetics due to multiple protein–silica contacts. However, during heating or pressurization scans, the infrared absorbance, A, was recorded for the preset temperature or pressure, after A had reached a constant value, which usually took 10–20 min. As can be seen from Fig. 5A and B, the temperature of unfolding (fraction folded = 0.5) of lysozyme is reduced from 78 to 63 ◦ C due to adsorption on silica particles, whereas that of RNase is reduced from 66 to 56 ◦ C. The pressures of unfolding are also reduced (fraction folded = 0.5 in Fig. 5C and D). In the case of lysozyme, adsorption leads to a reduction from 9.0 to 5.1 kbar, and in the case of RNase, we observe a drop from 7.7 to 4.7 kbar. Thus, the folded conformations of lysozyme and RNase are less stable against increased temperature and pressure in the adsorbed states of the proteins. For comparison, pressures of unfolding of 9.6 and 7.4 kbar are reported for free lysozyme [41] and free RNase [38], respectively, in good agreement with our values. Furthermore, from fitting Eqs. (5) and (6) to the measured infrared absorbance, A, the enthalpy change of unfolding, H, and the volume change of unfolding, V, are also obtained. These two thermodynamic parameters are proportional to the slopes at the inflection points in Fig. 5. Upon adsorption on silica particles, H of lysozyme is decreasing from 385 to 244 kJ mol−1 , and H of RNase is decreasing from 368 to 209 kJ mol−1 . This reduction can simply be explained by protein–silica interactions. H is the difference HU − HF , where the subscripts indicate the unfolded and the folded state of the protein. A stronger attractive protein–silica interaction of the unfolded state, as compared to the folded state, will reduce HU in a stronger way than HF , resulting in a decreased H value. The smaller H values observed in the adsorbed state of lysozyme and RNase are responsible for the broader temperature ranges of unfolding (Fig. 5A and B). Unfortunately, the V values, which can be derived from the fits in Fig. 5C and D, have a large error (about ±50%), because the precision of the pressure determination of the diamond anvil technique used here is rather low (about ±200 bar). Anyway, we find values of −21 mL mol−1 (free lysozyme), −24 mL mol−1 (adsorbed lysozyme), −123 mL mol−1 (free RNase), and −36 mL mol−1 (adsorbed RNase). In the literature, for example, values of −26 mL mol−1 [41] and −55 mL mol−1 [42] are reported for free lysozyme, and values of −45 mL mol−1 [40] and −15 mL mol−1 [34] are reported for free RNase illustrating the experimental difficulty to obtain precise V values. Recently, we have studied the pressure-induced unfolding of staphylococcal nuclease (SNase) by fluorescence spectroscopy. The volume change of unfolding, V, was found to be −73 mL mol−1 in the free state and −41 mL mol−1 , when SNase is adsorbed on silica particles [23]. This reduction of |V| (absolute value because of negative sign) upon protein adsorption has been explained by a negative volume change of adsorption [23]. A small negative volume change of adsorption has also been found for lysozyme at the silica–water interface using fluorescence spectroscopy and neutron reflectometry [43], suggesting an absolute volume change of unfolding, |V|, that is smaller for adsorbed lysozyme. Despite all these experimental challenges, there seems to be a trend that both H and |V| are smaller in the adsorbed state of a protein. And, smaller H and |V| values in the adsorbed state are equivalent to broader temperature and pressure ranges, where the protein unfolds.
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Fig. 5. Adsorption-induced destabilization of the folded conformation of lysozyme and RNase as probed by heating and pressurization. The absorbance of lysozyme at 1644 cm−1 and that of RNase at 1633 cm−1 has been measured and rescaled to cover the range from 0 to 1.
4. Conclusions
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All data obtained in this study point to a rather simple mechanism of adsorption-induced conformational changes of proteins. Whereas the unfolding pathway of a protein on the temperature or pressure axis remains largely unaltered upon adsorption, the folding stability is significantly reduced in the adsorbed state leading to broader unfolding ranges that are shifted to lower temperatures and pressures. Although we could observe this behavior for two proteins and two denaturing conditions, the adsorbent material is limited in this study to silica particles, which are characterized by a negative surface charge and a relatively high hydrophilicity. In this way, protein–interface interactions are dominated by electrostatic forces, and the hydration of the adsorbed protein is always similar to that of the free protein. These circumstances are probably favorable to preserve the conformational space of a protein. On the other hand, when proteins are adsorbed on hydrophobic surfaces, they may undergo a different unfolding pathway as compared to the solution, because conformational substates, which are highly unfavorable in aqueous solution, could be stabilized by hydrophobic interactions between the protein and the interface.
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Acknowledgment This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) (grant number Cz 77/3-1). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2015.03.043.
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