Rui
Lu
a,
Wen-Wei
Li
a,
Abraham
Katzir
c,
Yosef
Raichlin
d,
Han-Qing
Yu
*a and
Boris
Mizaikoff
*b
aDepartment of Chemistry, University of Science and Technology of China, Hefei 230026, P.R. China. E-mail: hqyu@ustc.edu.cn
bInstitute of Analytical and Bioanalytical Chemistry, University of Ulm, 89081 Ulm, Germany. E-mail: boris.mizaikoff@uni-ulm.de
cSchool of Physics, Tel-Aviv University, Tel-Aviv 69978, Israel
dDepartment of Applied Physics, Ariel University Center of Samaria, Ariel, Israel
First published on 12th November 2014
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy using a special waveguide based on a silver halide fiber was used for probing the heat-induced secondary structure and conformation changes of bovine serum albumin (BSA). From the secondary derivative and the curve fitting of the obtained ATR-FTIR spectra, the changes of the BSA secondary structure with temperature were clearly identified. Two different thermal denaturation temperature ranges (i.e., 50–52 and 80–82 °C, at which a change of the protein structure occurred) were determined, while only one denaturation temperature was previously identified via classical FTIR measurements. Additionally, taking advantage of two-dimensional correlation spectroscopy more detailed information on changes of the protein secondary structure was revealed. The developed method facilitates in situ, sensitive, and more in-depth probing of protein secondary structures, which represents a significant advancement compared to conventional characterization methods.
Such a heat-induced protein denaturation is detectable by various spectroscopic methods, including ultraviolet-visible (UV/Vis) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) methods in various wavelength regimes, fluorescence spectroscopy, Raman and Fourier transform infrared (FTIR) spectroscopy, and after crystallization via X-ray crystallography.5,6 Among these techniques, FTIR has gained increasing relevance, because the vibrational spectrum is selective in the absorption band frequency position, widths, and intensities in response to protein structural changes.7 The most distinct and characteristic FTIR bands of proteins are the amide signatures. In particular, the amide I band (1700–1600 cm−1), which convolutes the major structural domain elements of proteins including α helix, β sheet, β turn, random coil, and anti-parallel intermolecular β sheet,8 has frequently been used to reveal the secondary structure of a protein. For FTIR detection, only a small amount of protein sample (approximately 10–100 μg) is needed. Therefore, it offers a facile method to study heat-induced dynamics and associated secondary structure changes of BSA6,7 and other proteins in aqueous solution.2–4,9 However, conventional FTIR spectroscopy provides only limited detection accuracy and sensitivity for monitoring the protein denaturation processes. Among the most substantial challenges in this respect is the fact that the bands of individual domain elements constituting the secondary structure typically overlap with each other. As a consequence, a precise band assignment in the amide I region and a quantitative identification of the secondary structure become difficult. Therefore, to extract more useful and accurate information on the denaturation process, band deconvolution and assignment of FTIR spectra require further optimization.
A recent trend in this direction is applying attenuated total reflection (ATR) infrared spectroscopy, which enables the direct detection of analytes at solid/liquid interfaces. Silver halide fibers are increasingly adopted as a suitable waveguide material in ATR spectroscopy due to their broad infrared-transparent window and a finely tunable flexible geometry.10 Previously, planar-tapered silver halide fibers have been used to effectively detect specific molecular constituents,11–13 to simultaneously determine multiple volatile organics,14 or to analyze biomolecules.15 The usage of fiberoptic waveguides as active sensing elements facilitates an increased number of internal inflections such that the intensity of the evanescent field and the resulting analytical signal can be significantly enhanced. However, to date there has been no report demonstrating the utility of such miniaturized fiberoptic sensors for probing protein structures or for advancing 2D correlation spectroscopy.
Here, a planar-tapered silver halide fiber was utilized for the first time to characterize protein denaturation processes. The heat-induced denaturation process of BSA was successfully identified by ATR-FTIR spectroscopy along with optimized band assignments. For the band assignments, we have employed a curve fitting method to extract detailed relevant information of structural variations from the temperature-dependent spectra. For the band sequential order,16 we have introduced two-dimensional correlation spectroscopy (2DCoS)17–19 to explore the secondary structural changes of BSA in detail.
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Fig. 1 ATR-FTIR spectra (A) and second derivative spectra (B) of BSA in D2O as a function of temperature at 48, 50, 52, 68, 80, 82, and 88 °C, respectively (after smoothing and baseline correction). |
For both the FTIR spectra and the second derivative spectra, the most distinct changes were determined in two dedicated temperatures ranges, i.e., 50–52 °C and 80–82 °C. Specifically, when raising the temperature from 50 to 52 °C, the peak at 1662 cm−1 disappeared, while a new peak at 1680 cm−1 emerged. According to a previous study,7 these two peaks should be ascribed to the β-turn. Subsequently, when the temperature was increased from 80 to 82 °C, the 1662 cm−1 peak appeared again. Evidently, a substantial change of the protein structure must have occurred in these distinct temperature ranges. Another noticeable peak is at 1613 cm−1, whose intensity increased markedly with increasing temperature. Since this peak is mainly associated with the intermolecular β-sheet structure resulting from the aggregation,7 our results strongly imply an irreversible aggregation of the protein and formation of the β-sheet structures. Combining these results, it can be derived that protein aggregation occurred at 50–52 °C, during which the unfolding of BSA became irreversible after the formation of intermolecular β-sheets.
It should be noted here that two different thermal denaturation temperature ranges were directly and distinctly observed in our study, which is attributed to the application of the planar-tapered silver halide fibers during the ATR-FTIR analysis. Given the significantly enhanced signal-to-noise ratio in contrast to using conventional, i.e., macroscopic multi-reflection ATR crystals, these distinct denaturation ranges have been IR-spectroscopically visualized in such a pronounced fashion for the first time. Consequently, the developed measurement technique along with the obtained results present a substantial improvement compared to the conventional FTIR measurements, as further detailed below.
The fitting curves of the BSA spectra at 48 °C reveal 6 relevant bands (1613, 1632, 1642, 1662, 1670, and 1690 cm−1) (Fig. 2A). Here, the bands at 1613 cm−1 are assigned to heating-induced intermolecular β-sheet structures, while 1642 cm−1 reflects random coils, 1662, 1670 and 1690 cm−1 are attributed to β-turns.6,7 However, the assigning of the band at near 1632 cm−1 has been under controversy. Although it has been assigned to an intra-molecular β-sheet structure for many proteins,27 several other studies suggest that it could be associated with the short segment chains connecting α-helix segments for some other proteins.28,29 A later study convinces that for BSA this band is not ascribed to the intra-molecular β-sheet structure.7 Therefore, here we assign the peak at 1630 cm−1 to the short segment chains connecting α-helix segments. This interpretation also suggests that the secondary structures of BSA are more flexible and strongly exposed to the solvent.
The bands obtained after fitting the spectrum at 68 °C were located at 1608, 1613, 1620, 1632, 1650, 1662, 1670, and 1680 cm−1 (Fig. 2B). Compared with those at lower temperatures, two of the bands (1642 and 1690 cm−1) disappeared, while four new bands (1608, 1620, 1650, 1680 cm−1) were observed. These new bands are assigned to the vibration of some amino acid residues, intermolecular β-sheet structure, α-helix, and β-turn, respectively.6,7
When further elevating the temperature to 88 °C, also eight bands were obtained after deconvolution (Fig. 2C). It is interesting to note that the two disappeared bands (1642 and 1690 cm−1) emerged again. Five of the bands observed at 68 °C (1608, 1613, 1620, 1662, 1680 cm−1) remained, and one new band occurred at 1655 cm−1. This band is assigned to the α-helix content, possibly resulting from a shift of the 1650 cm−1 band in the medium temperature range. All determined FTIR band assignments are summarized in Table 1. Apparently, fitting the FTIR spectral data within the amide I region using Gaussian band profiles facilitated the detailed identification of an increased number of individual band components, and therefore the efficient extraction of significantly more relevant information on BSA secondary structure changes compared to previously reported conventional methods.7
Wavenumber/cm−1 | Assignment |
---|---|
1680–1690 | β-turn |
1662–1670 | β-turn |
1650–1655 | α-helix |
1642–1645 | Random coil |
1632–1638 | Short-segment chains connecting the α-helical segment |
1610–1620 | Intermolecular β-sheet |
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Fig. 3 IR absorbance (i.e., peak area) represented as a function of temperature at the fitted wavenumbers. |
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Fig. 4 Synchronous and asynchronous 2D ATR-FTIR correlation spectra constructed from 31 spectra recorded at 2 °C BSA during thermal transition from 30 to 90 °C. |
(v1, v2) | Φ (v1, v2) | Ψ (v1, v2) | Sequential order |
---|---|---|---|
(1610, 1642) | + | + | 1610 > 1642 |
(1642, 1686) | + | − | 1642 < 1686 |
From the signs of the synchronous cross-peaks at (1610, 1642) and (1642, 1686) (listed in Table 2), the ‘sequential order’ of the intensity changes of the three bands during the heating process was obtained as: 1610 > 1642 cm−1, 1642 < 1686 cm−1. Since the three bands represent intermolecular β-sheet (1610 cm−1), random coil (1642 cm−1), and β-turn (1686 cm−1), respectively, the band sequential orders obtained here thus imply that both the intermolecular β-sheet and β-turn are more sensitive than the random coil to temperature. Thus, they underwent the most significant changes at increasing temperature.
Thus, sensitive and detailed probing of the heat-induced denaturation process of BSA was achieved in our study, attributed to the use of ATR-FTIR spectroscopy based on planar-tapered silver halide waveguide elements along with optimized band assignments and the band sequential order. The use of spectral curve fitting and 2DCoS analysis offered additional detailed information on the secondary structural change orders and thermal dynamics of BSA, thereby greatly advancing the detection of BSA structural changes.
Compared with conventional FTIR measurements, the developed ATR-FTIR probe with planar silver halide fibers as waveguides not only enables in situ measurements, but also significantly reduces the background absorbance while increasing the targeted analytical signal. Thereby, significantly more sensitive and reliable probing of protein structures under complex environmental conditions is achieved. Several remarkable improvements of our methodology in comparison with previous researches are noticeable. Firstly, the analysis of the secondary derivative ATR-FTIR spectra within the characteristic amide I (1600–1700 cm−1) regime at different temperatures clearly revealed two distinct thermal denaturation ranges for BSA, at which a change of the protein structure occurred (Fig. 1B, 2 and 3). To date, however, only one temperature has been identified from the secondary derivative spectra obtained via conventional FTIR measurements within the same spectral range (1700–1600 cm−1),7 while for the first denaturation temperature an additional spectral regime was further needed. Secondly, with the relatively high resolution of spectra scanning, more individual components bands can be revealed compared to conventional FTIR measurements (Fig. 2). Lastly, ATR-FTIR exhibited significantly higher intensities of the individual components convoluted into the spectrum (Fig. 3). Thus, the developed methodology apparently reveals more detailed information on the protein structure changes compared to conventional techniques.
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