An instrument which combines Raman spectroscopy with dynamic light scattering (DLS) can be used to study lysozyme’s unfolding and refolding processes under thermal stress conditions. Through the combination of these complementary techniques, secondary and tertiary protein structural markers (Raman spectroscopy) along with size changes (DLS) can be utilized in order to gain a better understanding of the unfolding behavior of lysozyme and other proteins.
在这些实验研究中，注意骨骼（930厘米）-1- 950厘米- 1），酰胺I（1600厘米-1- 1700 cm-1）和酰胺III（1200厘米-1- 1300厘米-1) marker regions in the Raman spectra. Variations in these areas suggest changes in protein’s secondary structure. Moreover, changes in tertiary structure can be seen by analyzing tyrosine (850 cm-1) and tryptophan (1550 cm-1) bands in the Raman spectra。这些区域的变化表明侧链的局部疏水性或亲水环境的变化，这是典型的展开。使用DLS测量以分析溶液中热应激溶菌酶的尺寸变化。
随着蛋白质加热和聚合和unfold, it becomes larger. This increase in size as well as the changes in the protein structural markers in the Raman data, describes the lysozyme’s unfolding behavior.
Methods and Materials
A fiber-coupled Raman spectrometer with a Zetasizer Nano ZSP is integrated within Malvern Panalytical’ Zetasizer Helix (ZS Helix) to provide Raman (conformational stability) and DLS (colloidal stability) data in sequence on a single sample.
The Zetasizer Nano system combines non-invasive backscatter (NIBS) detector technology with electrophoretic (ELS), static (SLS) and dynamic (DLS) light scattering to quantify the hydrodynamic radius of proteins from 0.3 nm to 10 µm, at concentrations from 0.1 mg/mL to 100 mg/mL, or more. Raman spectra were then collected using 785 nm excitation (~280 mW) from 150 cm-1to 1925 cm-1在4厘米-1解析度。
Lysozyme from the white of a chicken egg was formulated at concentrations of 3mg/mL and 30 mg/mL in a pH 4 citric acid buffer. Next, sample aliquots (~120µL) were introduced into the sample compartment by means of a quartz cuvette with a 3 mm pathlength. The temperature of the sample compartment can be set from 0 °C to 90°C ± 0.1°C.
通过在低温下采用拉曼光谱和DLS测量来进行热跳跃实验，快速将样品室中的温度迅速增加到高温，进行另一个拉曼和DLS测量，然后将室内的温度返回到初始低温，在执行另一种之前Raman and DLS measurement.Thermal ramp studies were carried out collecting DLS and Raman data at a range of pre-defined 0.1°C to 2°C step-wise temperature increments.
Results and Discussion
Temperature Jump Studies
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图1。拉曼光谱（左）和DLS尺寸数据（右）用于使用3mg / ml（a）和30mg / ml（b）溶菌酶溶液的温度跳跃研究。所有光谱数据都是校正和归一化到PHE峰的高度的背景。
最初使用温度跳跃研究来研究热应力诱导的溶菌酶展开的可逆性（图1）。从热跳转实验中获得的拉曼和DLS数据的组合表明发生溶菌酶展开，并且这些变化可能是可逆的。
Both sets of parameters studied suggest that when the temperature is increased from 20°C (indicated by the black line) to 80°C (indicated by the red line), variations are seen in the structure of protein for 3mg/mL (Figure 1A, left) as well as 30 mg/mL (Figure 1B, left) solutions.
The Raman spectra revealed changes in both secondary and tertiary structural markers during heating. Upon reversing the temperature to 20°C (indicated by the gray line), the changes are also reversed. This suggests that structural changes can be reversed once the temperature is decreased.
Likewise, the DLS data obtained with Raman data from the lysozyme solutions demonstrate that when the temperature is raised from 20°C to 80°C, the protein’s size increases from ~4 nm to ~5 nm for the 3 mg/mL lysozyme solution (Figure 1A, right) and from ~4 nm to ~7 nm for the 30 mg/mL lysozyme solution (Figure 1B, right).
如预测，对于较高浓度的溶菌酶溶液，尺寸变化较大。这些尺寸的变化表明蛋白质在较高温度下开始展开。然而，不发生完全变性，因为这可能导致聚集。
For further analysis of the size and structural changes that lysozyme undergoes while subjected to thermal stress, temperature ramping studies were carried out.
Temperature Ramp Studies
Temperature ramp studies were carried out by collectingRaman and DLS dataat each 1°C increment between 20°C and 80°C, and repeating between 80°C and 20°C. The DLS and Raman spectral data from the thermal ramping studies suggest that as the temperature increases, there are size changes (Figure 2B) as well as secondary and tertiary structural changes (Figure 2A) in the lysozyme in solution.
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Figure 2.Raman and DLS data from a complete temperature ramp cycle with 30 mg/mL lysozyme sample.
After the temperature is decreased to the original temperature of 20°C (red circles), the size also goes back to the original size of ~4.5nm (Figure 2B). Further substantiation of this reversibility is provided by comparing the percent intensity distributions at 20°C (black line), 20°C (gray line) (Fig. 2B, inset), and 80°C (red line).
The Raman spectral data, together with the DLS data from the thermal ramping study, again indicate that size and structural changes occur at higher temperatures and these size and structural changes are reversible.
结论
Combining DLS and Raman spectroscopy into a single instrument is effective in studying the reversibility of the unfolding process in lysozyme while subjected to temperature changes at pH 4. The Raman and DLS results indicate that lysozyme was starting the unfolding process when the temperature was increased, as DLS data demonstrated that there was an increase in protein size, while Raman spectral data indicated that secondary and tertiary structural markers changed.
当温度降低到20℃的起始温度时，光谱和尺寸数据表明在较高温度下观察到的变化逆转，表明发生的任何发生的展开是可逆的。在热应力期间的两种蛋白质特征，结构和尺寸的同时监测在热应力期间提供了更全面的蛋白质行为的图像。
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