Article pubs.acs.org/ac
Sequential Order of the Secondary Structure Transitions of Proteins under External Perturbations: Regenerated Silk Fibroin under Thermal Treatment Zhipeng He,† Tingting Zhao,† Xiaofeng Zhou,† Zhao Liu,† and He Huang*,†,‡ †
Jiangsu Key Laboratory for the Design and Application of Advanced Functional Polymer, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China ‡ Zhangjiagang Institute of Industrial Technologies of Soochow University, Zhangjiagang, 215600, China S Supporting Information *
ABSTRACT: Whether the process of protein folding/unfolding is fully cooperative or it contains sequential elements has long been a fundamental issue in protein science. This issue seemingly became straightforward since the appearance of generalized two-dimensional (2D) correlation spectroscopy in 1990s, because 2D correlation analysis has been considered as a convenient and powerful analytical tool to determine the sequential order of events under external physical or chemical perturbations. In this work, the sequential order of the secondary structure transitions of regenerated silk fibroin under thermal treatment from 130 to 220 °C was first studied using generalized 2D correlation spectroscopy, but an apparently doubtful sequential order was obtained; β-sheet was the first one to change at low temperature, then the random coil, followed by the nonamide CO and, finally, the α-helix. A subsequent detailed in situ infrared spectral analysis showed that the main secondary structures of silk fibroin, including α-helix, β-turn, random coil and β-sheet (high-wavenumber component), all changed with a fully cooperative manner at a relatively low temperature of 150 °C. But the low-wavenumber component of β-sheet started to change at a higher temperature of 180 °C. Besides, it has also been found that, before 200 °C, the loss of α-helix and random coil was transformed into β-turn, β-sheet, and nonamide CO. After 200 °C, some β-turn structure was also disruptured and transformed into β-sheet and nonamide CO.
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spectra of Cro-V55C recorded over the temperature range of 20−95 °C. Based on observation of a number of distinctive features in the asynchronous 2D IR correlation plots and the result of spectral simulation, they concluded that 2D correlation analysis indeed provided evidence for a sequential formation of the stable intermediate. Peng et al.3 also employed generalized 2D correlation spectroscopy to study conformational transitions of regenerated silk fibroin when the film was heated from 130 to 220 °C or maintained at 180 °C for different time. According to the result of 2D correlation analysis on 1D infrared spectra, they concluded that random coil (1650 cm−1) and nonamide C O (1720 cm−1) changed prior to β-sheet (1630 cm−1) and αhelix (1660 cm−1). They thereby proposed that the sequential order of the response of secondary structures to temperature variation is as follows: the collapse of random coil and the oxidation of silk fibroin occurred first, followed by the increase or perfection of β-sheet and α-helix structures.
hether the process of protein folding/unfolding is fully cooperative or it contains sequential elements has long been a fundamental issue in protein science. It was not easy to address this question in the past, because probes used to monitor protein folding/unfolding phenomenon are not always sensitive enough to detect minor noncooperative events.1 This question, however, seems to be easily solved since the appearance of generalized two-dimensional (2D) correlation spectroscopy in 1990s, which was attributed to one major advantage of this analytical technique, sequential order determination of events via the straightforward “sequential order” rules of this technique.1,2 It has been generally believed that this technique provides a sensitive means of detecting a potential sequence of events in response to changes induced by external perturbing factors, such as temperature.1,3 As a result, almost all studies on protein folding/unfolding using generalized 2D correlation spectroscopy have concluded that the folding/unfolding of a protein is a process with sequential elements.1−17 Fabian et al.1 were among the pioneers who explored the protein folding/unfolding phenomenon with temperature variation using generalized 2D correlation spectroscopy. In their study,1 a 2D correlation analysis was performed on IR © 2017 American Chemical Society
Received: February 16, 2017 Accepted: April 13, 2017 Published: April 13, 2017 5534
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FTIR Measurement. The BaF2 window with a regenerated silk fibroin film was fixed on a horizontal heating accessory. After maintaining the sample at each temperature between 20 and 220 °C for 5 min, the FT-IR spectra with a temperature increment of 10 °C were collected by coadding 32 scans with a resolution of 2 cm−1 on a Nicolet 6700 FTIR spectrometer equipped with a DTGS detector. Data Processing. After an automatic baseline correction in the whole spectral range, the 1800−1580 cm−1 region of the FTIR spectra was truncated and subjected to a series of data management, including calculation of difference and second derivative spectra (with a Savitsky Golay derivative function), Fourier self-deconvolution (with a bandwidth of 80 cm−1 and enhancement factor of 3), and curve-fitting (with a Lorentzian/ Gaussian band shape, a moderate sensitivity and no baseline). All these data analyses were carried out using the OMNIC 8 software provided by the Thermofisher Scientific Company. 2D IR Correlation Analysis. The IR spectral range of 1800−1580 cm−1 was selected for 2D IR correlation analysis using the 2Dshige software, written at Kwansei Gakuin University. In the 2D correlation plots, unshaded regions indicate positive correlation intensities, and shaded ones are negative.
Conclusions such as above and many more from other researches2−17 on the sequential events of folding/unfolding process of proteins were directly based on the appearance of asynchronous correlation peak in the 2D correlation plot and the “sequential order” rules in generalized 2D correlation spectroscopy. But appearance of asynchronous correlation peak in the 2D correlation plot does not guarantee the existence of sequential order between two events. A simple nonlinear change in band intensity and factors such as peak shift, bandwidth change, baseline shift, and so on will lead to the creation of asynchronous correlation peak in the 2D correlation plot.18 In addition, our continuous work19−24 and that of other’s 25 demonstrate that sequential order of events determined by the “sequential order” rules of generalized 2D correlation spectroscopy may lead to ambiguous or even wrong conclusions, because the physical significance of the integrated/ overall sequential order (one event occurs prior/after another one) in generalized 2D correlation analysis was neither welldefined nor so meaningful in general situations, and the word “occur” used in the “sequential order” rules may easily give rise to ambiguity.24 In contrast to the integrated/overall sequential order, we proposed a chronological/local sequential order for nonperiodic changes in general situations and concluded that to judge whether one event occurs/happens before or after another one for two nonperiodic changes in general situations, the original spectral intensity changes must be verified to determine if a chronological/local sequential order exists between two events.19−24 Based on the above background, it may be important to reexamine the sequential order results of protein folding/ unfolding derived from generalized 2D correlation spectroscopy in response to external perturbations, such as temperature changes mentioned above.1,3 Compared to Cro-V55C, silk fibroin is a more common protein and easy to find. Therefore, in this work, the sequential order of the secondary structural changes of silk fibroin in its film which is under thermal treatment from 130 to 220 °C will be investigated, combining in situ 1D spectral analysis and 2D correlation analysis.
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RESULTS AND DISCUSSION 1D IR Spectra of Regenerated Silk Fibroin at Different Temperatures. A series of 1D IR spectrum of regenerated silk fibroin film in the range of 1800−1580 cm−1, taken under temperatures from 130 to 220 °C, were shown in Figure 1. This
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EXPERIMENTAL SECTION Materials. B. mori silk fiber was a product of Suzhou Suhao Biological Technology Co., Ltd. Chemical reagents, sodium carbonate, calcium chloride, ethanol, and so on, were purchased from Sinopharm Chemical Reagent (Shanghai, China) and used without any further purification. Sample Preparation. The B. mori silk fiber was first degummed 3 times, 30 min each time, in a 0.5% (w/w) Na2CO3 solution at 100 °C, then washed thoroughly with hot Na2CO3 solution for three times, followed by distilled water, and finally dried at 60 °C overnight. Subsequently, the degummed and dried silk fiber, i.e., silk fibroin, was dissolved in the mixture of CaCl2-EtOH-H2O (1:2:8, molar ratio) at 60 °C under constant stirring. This solution was dialyzed against deionized water using dialysis tubes with 8000 kDa (Sinopharm Chemical Reagent, China) for 3 days to remove the salt ions. After centrifugation and removal of impurities, the regenerated silk fibroin aqueous solution was concentrated to about 3% (w/ w) and stored in the fridge before use. A thin film of silk fibroin was obtained by casting the above regenerated silk fibroin aqueous solution onto a clean BaF2 window and dried slowly at 25 °C and under 50% relative humidity until the weight of the film did not change anymore. The thickness of the film was about 5 μm.
Figure 1. IR spectra of a regenerated silk fibroin film when heated from 130 to 220 °C.
figure is quite similar to Figure 1 in the literature,3 except that the spectra in this study are relatively smoother. The region of amide I was selected for investigation because the sub-bands under the amide I band profile have been generally accepted to be associated with different secondary structures of the protein. For example, the band located around 1630, 1650, and 1660 cm−1 were assigned to β-sheet, random coil, and α-helix structures,26−28 respectively. Figure 1 is dominated by an intensive band centered at 1655 cm−1 at low temperature, indicating that the secondary structure of the regenerated silk fibroin film is mainly composed of random coil and α-helix. With the increase of temperature, the band center shifts from 1655 cm−1 to higher wavenumbers and its intensity declines continuously, while the band broadens toward higher wavenumbers. At the same time, a broad new band centered around 1720 cm−1 appears and its intensity increases significantly with 5535
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Figure 2. Synchronous (A) and asynchronous (B) plots of regenerated silk fibroin film when heated from 130 to 220 °C with a contour level of 8.
different from that of B’s in the same group, as also found by some previously active practitionors of generalized 2D correlation spectroscopy. Comparing with the result in literature,3 we also found that the 1660 cm−1 band associated with α-helix structure and the 1630 cm−1 band associated with β-sheet structure (lowwavenumber component) were not resolved in this study. Instead, a band located around 1673 cm−1 associated with βturn structure and a band located around 1643 cm−1 associated with random coil structure were resolved, which are, however, not found in the literature.3 To check whether the missing of the 1660 and 1630 cm−1 bands was due to a low contour level of 8, 2D correlation analyses with higher contour levels of 16 and 32 were also carried out (see Figures S1 and S2 in Supporting Information), and the results were shown in Table 1.
increasing temperature. The appearance of a broad new band centered around 1720 cm−1, a nonamide CO band, was attributed to the thermal oxidation of the regenerated silk fibroin.3 The intensity decrease and shift toward higher wavenumber of the 1655 cm−1 band demonstrates that the amount of random coil and α-helix structures decrease with increasing temperature. Apparently, the 1D IR spectra of regenerated silk fibroin at different temperatures cannot provide more detailed information on the secondary structural changes of regenerated silk fibroin film under different temperatures, such as the sequential order of the secondary structural changes. 2D Correlation Analysis. It has been claimed that generalized 2D correlation spectroscopy has two major advantages over its 1D counterpart: spectral resolution enhancement and sequential order determination of events. Because of this, generalized 2D correlation spectroscopy has been widely used in the field of spectral analysis.2 Though we have tackled its potential pitfalls in preliminary studies as mentioned in the introduction section, 2D correlation analysis was also applied to the 1D IR spectra of regenerated silk fibroin at different temperatures to see whether there are any meaningful sequential orders among the secondary structures and whether the result is identical to that found in literature.3 The obtained 2D plots with a relatively low contour level of 8 were shown in Figure 2. Two bands at 1719 and 1651 cm−1 were observed in the synchronous plot. In the asynchronous plot, five bands around 1730, 1719, 1673, 1650, and 1643 cm−1 can be found, but the area between 1640 and 1600 cm−1 was not well resolved. In other words, at least a total number of five bands located around 1730, 1719, 1673, 1650, and 1643 cm−1 were resolved in this region of regenerated silk fibroin by 2D correlation analysis. Compared with the five bands, 1750, 1720, 1660, 1650, and 1630 cm−1, resolved in the literature,3 the total number of bands resolved is identical. But the band positions and shapes are largely different. Besides, the signs of the cross peaks in the asynchronous plot are also quite different from those in the literature.3 Such discrepancies, in fact, are a demonstration of another pitfall of generalized 2D correlation spectroscopy as we pointed out; the 2D correlation analysis result is largely dependent on the data sets used and very slightly different 1D IR data may lead to large difference in the 2D correlation analysis result.29 That is the reason that one research group’s conclusion based on 2D correlation analysis may be different from that of another one, and student A’s conclusion in one group may be
Table 1. Band Resolution from 2D Correlation Analyses with Different Contour Levels contour: 8 syn
asy
contour: 16 syn
asy
1602
1651
1719
1643 1650 1673 1719 1730
1651
1719
contour: 32 syn
asy
1602 1618 1643 1650 1673 1719 1730
1651
1719
1643 1650 1673 1714 1719 1730
It can be found that only one new weak band located around 1602 cm−1 was resolved in the synchronous plot when the contour level was increased to 16. When the contour level was 32, another new band around 1618 cm−1 was resolved in the synchronous plot, and one more new band around 1714 cm−1 was resolved in the asynchronous plot. The area between 1640 and 1600 cm−1 was still not well resolved for both higher contour levels and the two missing bands at 1660 and 1630 cm−1 were not found either. In summary, eight bands located at 1602, 1618, 1643, 1651, 1673, 1714, 1719, and 1730 cm−1 were resolved from the 2D correlation analyses of the 1D FTIR spectra of a regenerated silk fibroin film when heated from 130 to 220 °C (Table 2). This result seemingly verified the enhanced spectral resolution advantage of generalized 2D correlation spectroscopy. How5536
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Analytical Chemistry Table 2. Band Resolution and Assignments 2D 1602 1618 1643 1651 1673
SD
curve fitting
FSD
140
180
220
1611 1625
1611 1625
1629
1655 1678 1696
1655 1678 1696
1658 1678 1696
1714 1719 1730
140
180
220
1611 1625 1642 1656 1679 1696 1712
1611 1625 1642 1655 1679 1697 1714
1608 1626 1638 1657 1677 1697 1717
1727
1730
1731
1719
Table 3. Signs of the Cross Peaks and “Sequential Order” Analysis (ν1, ν2)
Syn
Asyn
sequential order
− − +
− + −
1720 > 1660 1650 > 1720 1630 > 1650
1611 1629 1646 1659 1679 1697
tyrosine side chain β-sheet random coil α-helix β-turn β-sheet
1724
nonamide CO vibration
mobile conformation than β-sheet, and it is more probable for β-sheet to change after random coil or simultaneously, rather than before it. The above result is also different from the conclusion of Peng et al.3 who found that the random coil (1650 cm−1) and the nonamide CO (1720 cm−1) due to thermal oxidation changed prior to β-sheet (1630 cm−1) and αhelix (1660 cm−1), when the regenerated silk fibroin film was heated from 130 to 220 °C or maintained at 180 °C for different time. In fact, the conclusion of Peng et al.3 may be questionable too, because it is inconceivable that the nonamide CO (1720 cm−1) due to thermal oxidation changed prior to β-sheet (1630 cm−1) and α-helix (1660 cm−1). Such a simple analysis demonstrates that the sequential order of events derived from the generalized 2D correlation spectroscopy may be wrong. This finding again verifies our previous assessment that it is not so meaningful to determine the integrated/overall sequential order of two events in general situations using generalized 2D correlation spectroscopy. Instead, the chronological/local “sequential order” must be determined.19−24 Therefore, it is important to analyze carefully the 1D IR spectra of regenerated silk fibroin film under thermal treatment to find out the real, if any, sequential order of band intensity changes existing among the secondary structural changes. Before doing so, the number of sub-bands under the amide I band profile must be sought out. Band Resolution and Assignment. Eight bands were resolved from the 2D correlation analyses of the 1D IR spectra of a regenerated silk fibroin film when heated from 130 to 220 °C and were shown in Table 2. In fact, second derivative (SD) and Fourier self-deconvolution (FSD) are the more commonly used classical tools for resolution of overlapped bands, though with their own pitfalls. Figure 3 presented the spectra of second
ever, are these bands resolved by 2D correlation analyses real? We will come back to this later on. In order to compare directly with the sequential order obtained in literature,3 identical cross peaks of (1720, 1660), (1720, 1650), and (1650, 1630) as in literature,3 rather than the cross peaks resolved in this study, were taken for sequential order determination, avoiding the potential confusion of using different cross peaks. It is applausible that though the bands 1660 and 1630 cm−1 were not well resolved in this study (Figure 2), the signs of the areas in which the cross peaks were located are clear, as shown in Table 3.
(1720, 1660) (1720, 1650) (1650, 1630)
assignment
From the signs of cross peaks shown in Table 3, it is easy to obtain the following sequence of intensity change of each subband under the band profile in the amide I region, according to “sequential order” rules:2 1630 > 1650 > 1720 > 1660 cm−1. This suggests that the β-sheet (1630 cm−1) was the first one to change at low temperature, then the random coil (1650 cm−1), followed by the nonamide CO due to thermal oxidation (1720 cm−1), and finally, the α-helix (1660 cm−1), that is, βsheet > random coil > nonamide CO > α-helix. This sequential order conclusion, however, is counterintuitive, because it is less probable for thermal oxidation to take place before the change of α-helix. Besides, random coil is a more
Figure 3. Second derivative (A) and Fourier self-deconvolution (B) analyses of the 1D IR spectra of regenerated silk fibroin film at 140, 180, and 220 °C. 5537
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On the contrast, the fit using the eight bands resolved from the second derivative and Fourier self-deconvolution analyses is perfect, though only one nonamide CO band was given the fitting data (Figure 4 and Table 2). This suggests that the band
derivative and Fourier self-deconvolution of three spectra shown in Figure 1 at 140, 180, and 220 °C, respectively. The bands resolved by second derivative and Fourier selfdeconvolution methods were also summarized in Table 2. Both the number of bands and the band positions resolved by a second derivative at 140 and 180 °C are identical. While, at 220 °C, the 1611 cm−1 band resolved at 140 and 180 °C was not detectable, a new band at 1719 cm−1 appeared. In addition, the two bands centered around 1625 and 1655 cm−1 at 140 and 180 °C were shifted to higher wavenumbers around 1629 and 1658 cm−1 when at 220 °C. In brief, a total number of six bands were obtained from second derivative analysis. Eight bands were resolved by Fourier self-deconvolution of the IR spectra of regenerated silk fibroin film at 140, 180, and 220 °C. The centers of five bands below 1700 cm−1 are almost identical at 140 and 180 °C, but the two above 1700 cm−1, 1712/1714 cm−1, 1727/1730 cm−1, are clearly different. More significant differences on band centers were found for the bands resolved at 220 °C. For example, the 1712 and 1714 cm−1 bands resolved at 140 and 180 °C, respectively, were found to appear at 1717 cm−1. The band resolution results from second derivative and Fourier self-deconvolution methodologies indicate that peak shift occurs while changing the temperature of the regenerated silk fibroin film, which is a common phenomenon in spectroscopic studies under thermal treatment of samples, including proteins. Combining the band resolution results from second derivative and Fourier self-deconvolution methodologies, it may be concluded that there are at least 8 bands in the region investigated (Table 2). In comparison with the eight bands resolved from the second derivative and Fourier self-deconvolution analyses, eight bands were also resolved by 2D correlation analyses of the 1D IR spectra of regenerated silk fibroin in the region of 1800−1580 cm−1. However, the 1696 cm−1 band resolved by second derivative and Fourier self- deconvolution analyses was not found from 2D correlation analyses. Besides, two bands around 1714 and 1719 cm−1 attributed to the nonamide CO stretching have been resolved by 2D correlation analysis. It is not safe to propose that there are another two nonamide CO stretching bands in addition to the 1730 cm−1 band. As evidenced in the Fourier self-deconvolution analysis, the 1717 cm−1 (or 1719 cm−1) band is a result of peak shift of the band centered around 1712 cm−1 (or 1714 cm−1) at 140 °C (or 180 °C). So, the splitting of the nonamide CO stretching band due to thermal oxidation of regenerated silk fibroin at high temperature (≥170 °C) in 2D correlation analysis is only a result of peak shift as discussed before.29 Furthermore, the centers of the two bands around 1602 and 1618 cm−1 are also different from those resolved by second derivative and Fourier self-deconvolution analyses. To further verify the number of bands and band centers in the region of 1740 and 1580 cm−1, the technique of curvefitting analysis was also adopted. Though the curve-fitting technique itself cannot tell exactly the number of sub-bands under a band profile, the curve-fitting result may suggest whether the number of sub-bands proposed is acceptable or not. Curve-fitting the IR spectrum of regenerated silk fibroin film at 140 °C, as an example, using the number of bands resolved from 2D correlation analyses is poor (see Figure S3 in Supporting Information), which indicates that the band resolution result by 2D correlation analyses is not acceptable.
Figure 4. Schematic of curve-fitting the IR spectrum of regenerated silk fibroin film at 140 °C using the eight sub-bands resolved from FSD and SD analyses.
resolution results from the second derivative and Fourier selfdeconvolution analyses is more reliable. It therefore can be concluded that the extra bands resolved by 2D correlation analysis over the second derivative or Fourier self-deconvolution analysis are not real, and the 2D correlation analysis does not exhibit any advantage over the classical band resolution techniques such as second derivative and Fourier selfdeconvolution. In the curve-fitting result (Figure 4 and Table 2), the 1724 cm−1 band is the nonamide CO due to thermal oxidation of regenerated silk fibroin at high temperature (≥170 °C),3 and the 1611 cm−1 band is associated with the side chain of the protein. Therefore, there are at least five sub-bands under the amide I band profile based on the second derivative and Fourier self-deconvolution analyses, and they can be easily assigned to the β-sheet (low wavenumber, 1629 cm−1), random coil (1646 cm−1), α-helix (1659 cm−1), β-turn (1679 cm−1), and β-sheet (high wavenumber, 1697 cm−1) structures, respectively (Table 2).26−28 Sequential Order of the Intensity Changes of the SubBands. Curve-fitting technique was used to assist band resolution in the above section. Curve-fitting is in fact more useful when quantitative analysis was needed. Following the procedure shown in Figure 4, all the 1D spectra in Figure 1 were curve-fitted and the intensity of each sub-band under the amide I profile was presented in Figure 5. Taking the error of analysis into consideration, it is straightforward to follow the intensity change of each subband in Figure 5, from top to bottom, with the variation of temperature. The intensity of the 1659 cm−1 band (α-helix) was observed to start decreasing very slightly after 150 °C but quickly after 180 °C and reached the minimum at 220 °C. The intensity of the 1679 cm−1 band (β-turn) was found to increase slightly after 150 °C but decrease rapidly after 200 °C. The intensity of the 1646 cm−1 band (random coil) remained unchanged until 150 °C, but decreased from 150 to 220 °C. The intensity of the 1697 cm−1 band (high-wavenumber component of β-sheet) started to increase after 150 °C and continued to 220 °C. The intensity of the 1611 cm−1 band (side chain) is so low that it looks unchanged during the whole course of heating. The 5538
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common sense. After a careful reexamination of the curvefitting data in Figure 5, we found that the intensity of the 1724 cm−1 band was not zero, though very close to zero, before 160 °C. This observation shows that the 1724 cm−1 band is probably not due to thermal oxidation of silk fibroin. Considering the complex hydrogen bonding interactions in proteins, this band (1724 cm−1) may be assignable to the socalled “free” CO stretching band, as found in hydrogenbonded polymer blends30 and the self-association of Nmethylacetamide (NMA).31 Naturally, the amount of “free” nonamide CO stretching band (1724 cm−1) may increase before the rising of the β-sheet (low-wavenumber component, 1629 cm−1). Temperature-Induced Secondary Structure Changes of Regenerated Silk Fibroin. In the above section, the sequential order of the secondary structures were studied by following the intensity changes of the sub-bands via curvefitting the IR spectra of regenerated silk fibroin film when heated from 130 to 220 °C. It is well-known, however, that the thickness of a sample film may change at higher temperatures due to thermal expansion. This implies that the sequential order result obtained from the band intensity changes via a curve-fitting procedure may be still questionable. To solve this problem, the area, rather than intensity, change of each subband under the amide I region, which indicates the relative content change of each secondary structure and may eliminate the influence of film thickness variation has been plotted against temperature, as shown in Figure 6 and Table 5.
Figure 5. Intensity change of each sub-band under the amide I band profile with temperature variation.
intensity of the 1629 cm−1 band (low-wavenumber component of β-sheet) did not change until 180 °C, but started to increase and reached the maximum at 220 °C. The intensity of the 1724 cm−1 band (nonamide CO) showed no noticeable change until 160 °C and continued to increase after 160 °C until the end. The temperature of each sub-band starting to change its intensity was summarized in Table 4. This result is apparently different from the sequential order derived from 2D correlation analysis (β-sheet > random coil > nonamide CO > α-helix). As discussed in the previous section, the sequential order derived from 2D correlation analysis is doubtful. The result obtained from curve-fitting the in situ IR spectra is now of rationality. During the course of heating, the intensity of 1611 cm−1 remained unchanged probably because side chain is not a constituent of the main secondary structures of the silk fibroin. No measurable intensity changes were found for all the bands resolved below 150 °C, indicating no secondary structure changes at such a relatively low temperature. Above 150 °C, the intensity of 1659 cm−1 band (α-helix), 1679 cm−1 (β-turn), 1646 cm−1 band (random coil), and 1697 cm−1 band (high-wavenumber component of βsheet) started to change at the same time, that is, the main secondary structures of silk fibroin, the α-helix, β-turn, random coil, and β-sheet (high-wavenumber component, 1697 cm−1), changed in a fully cooperative manner. Subsequently, the intensity of the 1724 cm−1 band (nonamide CO) started to increase at 160 °C, that is, the so-called nonamide CO started to grow at a higher temperature than the main secondary structures of silk fibroin. This is understandable because 1724 cm−1 band was assigned to the nonamide CO stretching due to thermal oxidation which would naturally occur at a higher temperature. It was a surprise, however, that the 1724 cm−1 band was not the last one to change its intensity. Instead, the intensity of the 1629 cm−1 band (β-sheet, lowwavenumber component) started to increase at an even higher temperature of 180 °C. This observation suggests that something might be wrong. When a regenerated silk fibroin film was heated from 130 to 220 °C, all secondary structures of silk fibroin should change prior to its thermal oxidation from a
Figure 6. Area change of each sub-band under the amide I band profile during the course of heating.
It is auspicious that the relative area change of each sub-band under the Amide I region is essentially the same as the intensity change. This indicates that the sample thickness in our experiment did not have any measurable change during the course of heating, which also verifies our conclusion on the sequential order derived from band intensity changes. It is now clear that there were no secondary structure changes below 150 °C. Above 150 °C, the main secondary structures of silk fibroin, including α-helix, β-turn, random coil and high-wavenumber
Table 4. Temperature of Each Sub-Band Starting to Change Its Intensitya band center (cm−1) starting temperature (°C)
1659 150
1679 150
1646 150
1697 150
1629 180
1611
1724 160
Now the following sequential order of band intensity changes is clear: 1659 cm−1 (α-helix) ≈ 1679 cm−1 (β-turn) ≈ 1646 cm−1 (random coil) ≈ 1697 cm−1 (high-wavenumber component of β-sheet) > 1724 cm−1 (nonamide CO) > 1629 cm−1 (low-wavenumber component of β-sheet). a
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Analytical Chemistry Table 5. Temperature of Each Sub-Band Starting to Change Its Area band center (cm−1) starting temperature (°C) a
1659 150
1679 150
1646 150
160 found in Figure 5 and Table 4.
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component of β-sheet, all changed at same time with a fully cooperative manner. Besides, the loss of α-helix and random coil was transformed into β-turn and β-sheet (high-wavenumber component). After 170 °C, the amount of “free” nonamide CO stretching band (1724 cm−1) increases rapidly and the low-wavenumber component of β-sheet (1629 cm−1) also increases fast at a higher temperature 180 °C. From this stage to 200 °C, in addition to β-turn and high-wavenumber component of β-sheet, the loss of α-helix and random coil were also transformed into low-wavenumber component of β-sheet (1629 cm−1) and nonamide CO (1724 cm−1). After 200 °C, the area of the 1679 cm−1 band (β-turn) began to decrease, suggesting that some β-turn structure was also disruptured at such a high temperature and, together with α-helix and random coil, was transformed into β-sheet (high- and low-wavenumber component) and nonamide CO (1724 cm−1). It is interesting to notice that the high-wavenumber component of β-sheet (1697 cm−1) is easier to form and less stable than the low-wavenumber component of β-sheet (1629 cm−1) under thermal treatment. The reason behind this is not clear at present.
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1697 150
1629 180
1611
1724 170a
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b00592. The synchronous (A) and asynchronous (B) plots with a contour level of 16. The synchronous (A) and asynchronous (B) plots with a contour level of 32. An example of curve-fitting using the number of bands resolved from 2D correlation analyses (PDF).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
He Huang: 0000-0002-6547-9193 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Science Foundation of China (NSFC, no. 51373115), the PhD Programs Foundation of Ministry of Education of China (20113201110004) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) for financial support of this work.
CONCLUSIONS
The sequential order of the secondary structure transitions of regenerated silk fibroin under thermal treatment was investigated by two-dimensional correlation analysis and in situ infrared spectral analysis. Two-dimensional correlation analysis revealed the following sequential order: β-sheet > random coil > nonamide CO > α-helix, that is, β-sheet (low-wavenumber component) was the first one to change at low temperature, then the random coil, followed by the nonamide CO, and finally the α-helix. This sequential order is beyond common sense and apparently doubtful. The sequential order obtained from careful in situ infrared spectral analysis is as follows: 1659 cm−1 (α-helix) ≈ 1679 cm−1 (β-turn) ≈ 1646 cm−1 (random coil) ≈ 1697 cm−1 (highwavenumber component of β-sheet) > 1724 cm−1 (nonamide CO) > 1629 cm−1 (low-wavenumber component of βsheet). This suggests that the main secondary structures of silk fibroin, including α-helix, β-turn, random coil, and β-sheet (high-wavenumber component), all changed with a fully cooperative manner at a relatively low temperature. Sequential events happened at higher temperatures when the nonamide CO (1724 cm−1) and β-sheet (low-wavenumber component, 1629 cm−1) started to change. It has also been found from in situ infrared spectral analysis that, in the process of temperature elevation, the loss of α-helix and random coil was transformed into β-turn, β-sheet, and nonamide CO before 200 °C. After 200 °C, some β-turn structure was also disruptured and transformed into β-sheet and nonamide CO.
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