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建立人际资源圈溶菌酶结构改变尿素浓度和pH值的
2023-09-03 来源: 类别: Paper范文
Abstract
In this experiment, we mainly focus on the fluorescence intensity and molar ellipticity of lysozyme at urea concentrations ranging from 0 to 8 M. With the fluorescence intensity spectra, we study lysozyme structural alteration under the different conditions of urea concentration and pH and determine the approximate range of pH causing conformational transition. Also, by the CD spectra, we try to determine lysozyme secondary structure under the impact of urea concentration, pH and temperature. Through the experiment, we expect to get the result that the maximum fluorescence intensity wavelength undergoes red-shift at pH ranging from 2 to 3 and at urea concentration 3 M and above. As for CD spectra observation, it is an effective method which is consistent with the fluorescence measurements. Undergoing a structural transition at pH ranging from 2 to 3, lysozyme at higher temperature and higher urea concentration will have a structural transition at higher pH as well. And the midpoint transition pH of lysozyme is plotted as a function of urea concentration and temperature In addition, based on the result we observed from fluorescence intensity measurements and CD spectra, we create a phase diagram of lysozyme in the following parts, which allows us to determine whether lysozyme is retain in native state or in denatured state under different conditions.
Introduction
Proteins are large and versatile molecules that contain varieties of cellular functions. They are linear polymers made of amino acid residues. Many functional groups in protein, like methyl, carboxyl, and amino groups, contribute to protein’s diverse functions. For example, the protein may undergo conformational changes to form an active site for ligand binding when it performs enzyme functions. Proteine’s structure is very sensitive to salt, denaturants, and temperature and their folding/unfolding equilibrium can be grossly affected by the aqueous environment. Protein function can change because of the close relationship between its function and structure. Therefore, understanding protein structural alteration is very important in studying protein function.
Osmolytes are small organic molecules that stabilize or destabilize the folded or unfolded state of protein. TMAO, as an osmolytes, stabilizes proteins against pressure denaturation in deep-sea animals, while urea, as one of the most common cosolvents, acts opposite to TMAO. Protein denaturation can be explained by two mechanisms of the indirect and direct mechanism. The first mechanism postulates that urea causes the denaturation of proteins by altering the water structure. Water with altered structure interacts more with the hydrophobic group in protein. Without the addition of urea, the hydrophobic group in protein will tend to aggregate together to form stable structures in aqueous environments, which is known as hydrophobic effect. As a major driving force of protein folding, this effect is weakened by the addition of urea. On the other hand, the second mechanism implies the existence of direct Van der Waals or hydrogen bonding or other electrostatic interactions between urea and protein groups. Urea unfolds proteins by forming hydrogen bonds with the protein backbone. By competing with the intra-backbone hydrogen bonding that stabilizes the protein secondary structure, urea causes protein unfolding.^4
By modulating electrostatic interactions between amino acids, the solution of environmental pH can also influence the protein structure largely. The ionization of carboxyl and amino groups in protein are affected by pH and they can be protonated or deprotonated depending on pH. Meanwhile, protonation or deprotonation of some of these groups will alter the electrostatic interaction between amino acids. Addition of acids will protonate carboxyls, and make the protein mostly positively charged.^2 These positive charged groups will repel each other and lead to an extended conformation or unfolded protein.^2 The repulsion force generated will contribute to protein unfolding, but the hydrophobic effect will lead to protein folding. Hydrophobic groups tend to aggregate in polar environment to minimize the surface contact area with water. Plus, The statistical mechanical theory of protein stability also implies that the balance among the hydrophobic effect, the electrostatic effect, pH, and ionic strength determines the protein stability.^1 In other words, acid denaturation of protein is governed by the hydrophobic and the intramolecular charge-charge repulsion.^2
Fluorescence spectroscopy is one of the most useful techniques that can be used to detect protein structural changes of protein. Electrons in protein molecules get excited by absorbing a photon. After the electron jumps to an excited state, it will lose some energy due to the collision with other molecules and it will reach a lowest excited state. Then the electron will get back to the ground state and emit a visible photon at the same time. Emission spectra provide a sensitive mean to detect protein conformation. Fluorescence from tryptophan is very sensitive to its microenvironment. Following urea-induced denaturation of protein, the environment of tryptophan may change due to protein structural alteration. The tryptophan residue will be exposed to the aqueous environment instead of the hydrophobic protein interior. The maximum fluorescence will be observed at a higher wavelength, and red-shifted emission spectrum will be observed. Briefly, a red shift of emission spectrum will be observed upon exposure of an originally buried Trptpphan to a more polar environment. The blue shifts will occur when an originally buried tryptophan emerges in the surfactant vesicle or hydrophobic core.^6Water may contribute to the wavelength shift, but they usually dominate the mechanism of shift when charged groups stay close to Trp.^5
Circular dichroism is an effective technique to be widely used to study protein structure. The polarized light consists of two circular polarized components with the same magnitude but different rotation directions left and right. When chiral molecules interact with circular polarized lights, they will be absorbed to different extents. The CD instruments measure the difference in absorption. The differential is reported in terms of ellipticity in degrees.^8 CD signal will only be generated at chiral centers that are sensitive to their microenvironment. Thus, the spectral bands are easily distinguished between different structures. This feature helps CD to monitor the conformational changes in protein and determine the protein secondary and tertiary structure. UV CD spectra associated with various types of protein secondary structures such as, α-helix, β-sheet, and random coil. As is taken an example, CD was used to detect the protein transition between helical hairpin and coiled-coil.^8 In addition, CD can also be used to determine the extent of the protein structural change and the kinetics of this change.^8
Lysozymes, known as N-acetylmuramine glycanhydrolase, is a small globular protein. It can destroy peptidoglycan in bacteria walls by catalyzing the hydrolysis of beta glycosidic.^13 Many researchers have studied lysozymes for a long time. They provide us with good understanding of protein stability, solubility and conformational changes under the presence of the cosolvents. Apart from cosolvents or osmolytes, changes in environmental pH and temperature will give rise to its denaturation.
Materials and method
Materials. Urea, protein hen egg lysozyme and NaCl were purchased from Sigma Chemical (St.Louis MO). To ensure purity, lysozyme was dialyzed against distilled water first to remove any suspended particles and salts. Urea and NaCl were used without further purification.
Solution preparation. 0-8 M urea buffers were prepared first by adding calculated amount of urea and pre-estimated amounts of water together.
(1)
where C is the molar concentration, is the density of water, the is the density of urea at desired concentration, is the molecular weight of urea, is the mass of urea, and is the mass of water.
For each urea concentration, its density was measured by the densemeter (DMA-60, Anton Paar, Austria).
Spectroscopic measurements. The concentrations of lysozyme solution at all urea concentrations were measured by UV spectrophotometrically at. The extinction coefficients for each urea concentration was obtained from a presiousarticle Based on Beer-Lambert’s Law, the concentration can be determined by inversing the following equation.
(2)
where c is the molar concentration, l is the cell pathlength, is the extinction coefficient, and A is the light absorbance measured from UV spectroscopy. The protein solution concentrations were around for each urea solution.
pH titration. For each urea concentration, 1.5 mL of lysozyme solution was titrated with three different HCl stocks of various pH. The pH of lysozyme was measured by pH meter initially. By titrating different HCl stocks, the pH of lysozyme solution was decreased to ~1.5. The pH value and temperature of lysozyme solution after each addition of HCl stock were recorded.
Fluorescence titration. Fluorescence intensity measurements were performed at 25using an Aviv model ATF 105 spectofluorometer (Aviv Associates, Lakewood, NJ). The fluorescence intensity of lysozyme solution after each addition of HCl was measured by fluorescence spectroscopy. The titration profiles were measured by the incremental addition of aliquots of HCl to a 1cm path length cell containing 1.5 mL of lysozyme solution. Scans have been repeated three times to ensure the accuracy. The protein sample are exited at 295nm, the emission intensity were collected at 345nm through a monochoromator. For both pH titration and fluorescence titration, two trials were performed. The second trial was used to fill the gap between pH values and to make a smooth and accurate plot of pH versus intensity.
Circular dichroism. The CD spectra were recorded at 25 using an AVIV model 60 DS spectrophlarimeter (Aviv Associates, Lakewood, NJ). CD titration profiles were measured by incrementally adding HCl stocks each time to an optical cell containing 0.4mL of lysozyme. A 1mm cell was used for the CD measurements. The ellipticity was collected through wavelength of 210nm to 260nm.
Result
Figure 1. The urea density at different molar concentration.
As is shown above, the urea density increases with the growth of the molar concentration. Thus, we can use the linear as a calibration curve to calculate the density at a desired urea molar concentration.
Figure 2. Fluorescence spectra of lysozyme with 5M urea at acidic pH from 7.61-1.90.
Figure 2 tells that the fluorescence intensity of lysozyme presents a red shift at pH 2.93. Combined with Figure 1, the protein exhibits a maximum emission () wavelength of around 345 nm at acidic pH around 2.93. The shifts to around 358nm at pH 2.93 and begins to decline. The fluorescence intensity of lysozyme decreases gradually from ~16200 to ~14500 before pH reaches 2.93, while the intensity increases to ~18000 and the wavelength slightly shifts to right at pH 2.93. When the pH further decreases, the intensity significantly increases to around 29200, and the wavelength reaches around 358 nm. Then, from the graph above, we can see that the intensity gradually decreases again to 23600 as lysozyme titrated with more HCl buffer.
Figure 3. Fluorescence spectra of lysozyme with 3M urea at acidic pH from 7.07-1.67.
Likewise, Figure 3 shows that a relatively small red-shift occurred at acidic pH above 2.59. Lysozyme remains the same of around 345 nm before the pH decreased to 2.59, but an increase in was observed at acidic pH 2.59 or lower pH. Compared with Figure 2, the lysozyme with 3M urea could be seen to exhibit a smaller red shift in fluorescence intensity at a lower pH related to lysozyme with 5M urea. These two figures are examples that lysozyme at higher urea concentrations has a larger red shift at higher pH environment. The red shift appears at 3M or higher urea concentration, while lysozyme with urea concentration below 3M does not exhibit a shift in fluorescence intensity. The change in fluorescence maximum emission wavelength indicates a change in lysozyme structure, which suggests that no protein structure change at lower urea concentration even with the inducing of acidic pH. In addition, with the increase of the urea concentration from 0M to 8M, it becomes more difficult to decline pH of lysozyme solution down to around 1.5.
Figure 4. The lysozyme maximum intensity wavelength against aqueous environmental pH.
Figure 4 suggests that the wavelength increased with the decrease of environmental pH. The in the unfolded condition ranges from 340 to 345, while the shifts to 355-359 at low pH with the increase of urea concentration.
Figure 5. CD spectra of lysozyme solution with 3M urea.
Figure 5 shows that at pH 7.2, lysozyme exhibits a significant strucutre and there is a slight transition around pH 2.5. Also, upon a decrease in pH, the molar ellipticity only increases slightly, indicating that lysozyme was not fully denatured. Part of lysozyme may retain in structure.
Figure 6. CD spectra of lysozyme solution at 8M urea.
Figure 6 presents clear evidence for lysozyme unfolding. It can be seen that lysozyme retains before pH reaches around 3.5. The CD spectra at pH 2.24 is consistent with a random coil structure of lysozyme, but the molar ellipticity changes from -120000 to around -50000 and the change of relatively larger protein structure came into being at 8M urea than 3M urea.
Figure 7. Molar ellipticity of lysozyme with urea concentration from 0M to 8M at acidic pH 1.25-8.22.
Figure 7 shows the CD spectra of lysozyme at acidic pH of urea concentration from 0-8M. It tells that the molar ellipticity curves almost overlap above pH 3.5 without the effect of lysozyme concentration. Below pH 3.5, the lysozyme with 8M urea buffer has a pH and urea induced transition and the molar ellipticity gradually decreases with the decrease of urea concentration. Just slight or almost no transition could be seen in the lysozyme solutions of 0, 1 and 2 M urea buffer. But it could be seen that the transition midpoint shifts toward higher pH as the concentration of urea increases.
Figure 8. Molar ellipticity of lysozyme at 2 M urea concentration at 221 nm.
Figure 9. Molar ellipticity of lysozyme at 8M urea concentration at 221 nm.
Figure 9 tells that the molar ellipticity at 221 nm shifts slightly towards higher pH as temperature increases. Following the sigmoidal profile, the post denaturation baseline presents the completion of lysozyme denaturation and the lower baseline indicates that lysozyme retains in folded state above pH 3.5. Besides, a slight shift could be found in transition midpoint toward higher pH as the temperature increases.
Figure 10. Phase diagram of lysozyme denaturation. Midpoint transition pH is plotted as a function of urea concentration and temperature.
Figure 10 presents that the pH transition midpoint of lysozyme at 45 is generally higher than that at lower temperature. The blue linear is the steepest among all the three lines, while lines of lysozyme denaturation at 35 and 25 overlaps.
Discussion
Fluorescence intensity analysis. Figure 2 shows an increase in fluorescence intensity of lysozyme at 5M urea concentration around pH 2.93. This red shift may be attributed to a change in lysozyme structure. More specifically, the microenvironment of tryptophan (Trp) residues in lysozyme may change due to the cosolvent-protein interaction or the acid-induced denaturation. Exposure of a buried Trp to solvent results in a red shift of . The Trp residues in folded lysozyme present the maximum emission around 345 nm and the shift in to around 355 nm indicates the exposure of aromatic ring in Trp residue to aqueous environmental.^11 As for Figure, it tells that of lysozyme at 3M urea concentration has a smaller red-shift at pH around 2.59. Comparing Figure 2 with Figure 3, we can conclude that urea concentration plays an important role on lysozyme denaturation. As urea concentration increases, we can see a larger red shift and higher fluorescence intensity. In Figure 4, is plotted as a function of pH and urea concentration. When urea concentration increases, shifts to higher wavelength. Thus, urea concentration and lysozyme stability is found to have a close relationship, because urea arouses the denaturation of lysozyme by perturbing the intramolecular hydrogen bonding and the hydrophobic interaction between lysozyme molecules.^11 Also, it is found that higher urea concentration can lead to lysozyme less stable and easier to unfold.^12 Moreover, fluorescence intensity can become higher with the appearance of the transition, indicating a greater extent of unfolded lysozyme.
CD spectra analysis. Through the measurement of fluorescence intensity, lysozyme is found to undergo a conformational transition around pH 2.5-3. Our CD spectra provide more details about the change of protein secondary structure influenced by the urea concentration and acidic pH environment. Different structure will generate distinct CD spectra, so a small shift in molar ellipticity curve in Figure 6 tells us that lysozyme with 3 M urea concentration will have small conformational changes under the acidic conditions. And we can say that most of the lysozyme at 3 M urea retains some α-helix structure. Figure 7, meanwhile, presents a clear transition between α-helix and random coil. Therefore, a conclusion is drawn that lysozyme at higher urea concentration shifts to unfolded state more than that at lower uran concentration, witnessed from both Figure 6 and Figure 7.
Besides, from Figure 8 and Figure 9 above, we can find that temperature has an influence on protein unfolding: as temperature increases, the conformational transition of lysozyme comes into being at higher pH and lower urea concentration. At 45℃, the conformational transition of lysozyme at 8 M urea concentration occurs at pH around 4. Likewise, at the same temperature, part of lysozyme at 2 M urea concentration undergoes structure transition and the other part retains in strucutre. These two figures suggest that lysozyme is less stable and easier to change its conformation at higher temperature, whose reason is that higher temperature makes protein structure less stable. On the other hand, when temperature increases, the free energy required to unfold lysozyme decreases. Lysozyme retains its folded state, but it becomes less stable and is easier to overcome the intramolecular interaction between hydrophobic groups in lysozyme. Therefore, lysozyme is easier to unfold under the influence of urea and pH environment. And no structural transition occurs over the urea concentration range 0 to 2M except for lysozyme at 45.
Phase diagram analysis. As is shown in Figure 10, there are phase boundaries between lysozyme folding and unfolding phases. Area above or below boundaries is defined as the native state or the denatured state respectively. All three boundaries in the phase diagram of lysozyme have the positive slope, that is, the higher the urea concentration and the midpoint transition pH are, the more easily a conformational transition can occur. In addition, lower pH is required to denature 50 % of lysozyme at 25℃ and 35℃.
During the first stage of study, we have collected the fluorescence intensity and CD spectra at 0-8M urea concentration and constructed the phase diagram of lysozyme denaturation. Based on the results, subsequent experiments, including volumetric measurements of lysozyme structural transition, will be carried out in the second stage of study to further evaluate lysozyme structural transition.
Conclusion
By measuring the fluorescence intensity of lysozyme at urea concentration at range from 0 to 8 M, we suggest that shifts toward higher wavelength at pH ranging from 2 to 3 over the range of urea concentrations 3 to 8 M. As the urea concentration increases, will have a larger shift at lower pH. Meanwhile, through recording CD spectra, we ensure that lysozyme undergoes different structural alterations during the experiment, which can tell the secondary structure of lysozyme under the effect of pH and the presence of urea at three different temperatures. With our efforts of experiment and observations, we find that the results of CD measurements confirm to the fluorescence measurements and no structural transition occurs over the urea concentration range 0 to 2M except for lysozyme at 45. Lysozyme becomes less stable and easier to undergo a conformational change at higher temperature. Thus, the phase diagram shows lysozyme native/denatured state at different conditions, which better represents the relationship between protein stability and pH, urea concentration and temperature.
References
1.Darwin O., Ken A（1991）. The three states of globular proteins: acid denaturation. Biopolymers. Vol31, 1631-1649
2.Anthonyl, L. F., Linda, J., Yuji, G., Takuzo K., ﹠ Dabuek R.(1994). Claasification of acid denaturation of proteins: intermediates and unfolded. Biochemistry. Vol33. 12504-12511
3.John, W. K. (2013). The Rules of Protein Structure. Kimball's Biology Pages. Website. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DenaturingProtein.html
4.Deepak, R., Canchi ﹠ Angel E. G. (2013). Coslvent Effects on Protein Stability. The Annual Review of Physocal Chemistry. Vol64. 273-293
5.James, T. V., ﹠ Patrik R. C. (2001). Mechanisms of Tryptophan Fluorescence Shifts in Proteins. Biophysical Journal. Vol 80. 2093-2109
6.Gregory, A. Caputo, Erwin L. (2003). Cumulative Effects of Amino Acid Substitutions and Hydrophobic Mismatch upon the Transmembrane Stability and Conformation of Hydrophobic R-Helices. Biochemistry.Vol.42. 3275-3285
7.J. B. Alexander R., William R., Madeline, A. S. Intrinsic Fluorescence in Protein Structure Analysis. Methods in Protein Structure and Stability Analysis. Chapter 2.1. 55-72
8.Sharon M. K., Thomas J. J., Micholas C. P. (2005). How to study proteins by circular dichroism. Biochimica et Biophysica Acta. Vol1751. 119-139
9.Ikbae, S., Yuen, L. S., David N. D., and Tigrin V. C. (2012).Volumetric Characterization of Tri-N-acetylglycosamine Binding to Lysozyme. Biochemistry. Vol51. 5784-5790
10.Serge, N. T., Guifu X. (2002). Preferential interations of urea with lysozyme and their linkage to protein denaturation. Biophysical Chemistry. Vol105. 421-448
11.Alexander P. D. (2012). Quantum Mechanical- Classical Mechanical Methods for Solvatochromism and Electrochromism Predictions Chapter 2.3 Analysis Tools. (2010). 315-316. Advanced Fluorescence Reporters in Chemistry and Biology I : Fundamentals and Molecular Design. New York. Springer Heidelberg Dordrecht
12.John. A. S. (1994). The thermodynamics of Solvent Exchange. Biopolymers. Vol 34. 1015-1026
In this experiment, we mainly focus on the fluorescence intensity and molar ellipticity of lysozyme at urea concentrations ranging from 0 to 8 M. With the fluorescence intensity spectra, we study lysozyme structural alteration under the different conditions of urea concentration and pH and determine the approximate range of pH causing conformational transition. Also, by the CD spectra, we try to determine lysozyme secondary structure under the impact of urea concentration, pH and temperature. Through the experiment, we expect to get the result that the maximum fluorescence intensity wavelength undergoes red-shift at pH ranging from 2 to 3 and at urea concentration 3 M and above. As for CD spectra observation, it is an effective method which is consistent with the fluorescence measurements. Undergoing a structural transition at pH ranging from 2 to 3, lysozyme at higher temperature and higher urea concentration will have a structural transition at higher pH as well. And the midpoint transition pH of lysozyme is plotted as a function of urea concentration and temperature In addition, based on the result we observed from fluorescence intensity measurements and CD spectra, we create a phase diagram of lysozyme in the following parts, which allows us to determine whether lysozyme is retain in native state or in denatured state under different conditions.
Introduction
Proteins are large and versatile molecules that contain varieties of cellular functions. They are linear polymers made of amino acid residues. Many functional groups in protein, like methyl, carboxyl, and amino groups, contribute to protein’s diverse functions. For example, the protein may undergo conformational changes to form an active site for ligand binding when it performs enzyme functions. Proteine’s structure is very sensitive to salt, denaturants, and temperature and their folding/unfolding equilibrium can be grossly affected by the aqueous environment. Protein function can change because of the close relationship between its function and structure. Therefore, understanding protein structural alteration is very important in studying protein function.
Osmolytes are small organic molecules that stabilize or destabilize the folded or unfolded state of protein. TMAO, as an osmolytes, stabilizes proteins against pressure denaturation in deep-sea animals, while urea, as one of the most common cosolvents, acts opposite to TMAO. Protein denaturation can be explained by two mechanisms of the indirect and direct mechanism. The first mechanism postulates that urea causes the denaturation of proteins by altering the water structure. Water with altered structure interacts more with the hydrophobic group in protein. Without the addition of urea, the hydrophobic group in protein will tend to aggregate together to form stable structures in aqueous environments, which is known as hydrophobic effect. As a major driving force of protein folding, this effect is weakened by the addition of urea. On the other hand, the second mechanism implies the existence of direct Van der Waals or hydrogen bonding or other electrostatic interactions between urea and protein groups. Urea unfolds proteins by forming hydrogen bonds with the protein backbone. By competing with the intra-backbone hydrogen bonding that stabilizes the protein secondary structure, urea causes protein unfolding.^4
By modulating electrostatic interactions between amino acids, the solution of environmental pH can also influence the protein structure largely. The ionization of carboxyl and amino groups in protein are affected by pH and they can be protonated or deprotonated depending on pH. Meanwhile, protonation or deprotonation of some of these groups will alter the electrostatic interaction between amino acids. Addition of acids will protonate carboxyls, and make the protein mostly positively charged.^2 These positive charged groups will repel each other and lead to an extended conformation or unfolded protein.^2 The repulsion force generated will contribute to protein unfolding, but the hydrophobic effect will lead to protein folding. Hydrophobic groups tend to aggregate in polar environment to minimize the surface contact area with water. Plus, The statistical mechanical theory of protein stability also implies that the balance among the hydrophobic effect, the electrostatic effect, pH, and ionic strength determines the protein stability.^1 In other words, acid denaturation of protein is governed by the hydrophobic and the intramolecular charge-charge repulsion.^2
Fluorescence spectroscopy is one of the most useful techniques that can be used to detect protein structural changes of protein. Electrons in protein molecules get excited by absorbing a photon. After the electron jumps to an excited state, it will lose some energy due to the collision with other molecules and it will reach a lowest excited state. Then the electron will get back to the ground state and emit a visible photon at the same time. Emission spectra provide a sensitive mean to detect protein conformation. Fluorescence from tryptophan is very sensitive to its microenvironment. Following urea-induced denaturation of protein, the environment of tryptophan may change due to protein structural alteration. The tryptophan residue will be exposed to the aqueous environment instead of the hydrophobic protein interior. The maximum fluorescence will be observed at a higher wavelength, and red-shifted emission spectrum will be observed. Briefly, a red shift of emission spectrum will be observed upon exposure of an originally buried Trptpphan to a more polar environment. The blue shifts will occur when an originally buried tryptophan emerges in the surfactant vesicle or hydrophobic core.^6Water may contribute to the wavelength shift, but they usually dominate the mechanism of shift when charged groups stay close to Trp.^5
Circular dichroism is an effective technique to be widely used to study protein structure. The polarized light consists of two circular polarized components with the same magnitude but different rotation directions left and right. When chiral molecules interact with circular polarized lights, they will be absorbed to different extents. The CD instruments measure the difference in absorption. The differential is reported in terms of ellipticity in degrees.^8 CD signal will only be generated at chiral centers that are sensitive to their microenvironment. Thus, the spectral bands are easily distinguished between different structures. This feature helps CD to monitor the conformational changes in protein and determine the protein secondary and tertiary structure. UV CD spectra associated with various types of protein secondary structures such as, α-helix, β-sheet, and random coil. As is taken an example, CD was used to detect the protein transition between helical hairpin and coiled-coil.^8 In addition, CD can also be used to determine the extent of the protein structural change and the kinetics of this change.^8
Lysozymes, known as N-acetylmuramine glycanhydrolase, is a small globular protein. It can destroy peptidoglycan in bacteria walls by catalyzing the hydrolysis of beta glycosidic.^13 Many researchers have studied lysozymes for a long time. They provide us with good understanding of protein stability, solubility and conformational changes under the presence of the cosolvents. Apart from cosolvents or osmolytes, changes in environmental pH and temperature will give rise to its denaturation.
Materials and method
Materials. Urea, protein hen egg lysozyme and NaCl were purchased from Sigma Chemical (St.Louis MO). To ensure purity, lysozyme was dialyzed against distilled water first to remove any suspended particles and salts. Urea and NaCl were used without further purification.
Solution preparation. 0-8 M urea buffers were prepared first by adding calculated amount of urea and pre-estimated amounts of water together.
(1)
where C is the molar concentration, is the density of water, the is the density of urea at desired concentration, is the molecular weight of urea, is the mass of urea, and is the mass of water.
For each urea concentration, its density was measured by the densemeter (DMA-60, Anton Paar, Austria).
Spectroscopic measurements. The concentrations of lysozyme solution at all urea concentrations were measured by UV spectrophotometrically at. The extinction coefficients for each urea concentration was obtained from a presiousarticle Based on Beer-Lambert’s Law, the concentration can be determined by inversing the following equation.
(2)
where c is the molar concentration, l is the cell pathlength, is the extinction coefficient, and A is the light absorbance measured from UV spectroscopy. The protein solution concentrations were around for each urea solution.
pH titration. For each urea concentration, 1.5 mL of lysozyme solution was titrated with three different HCl stocks of various pH. The pH of lysozyme was measured by pH meter initially. By titrating different HCl stocks, the pH of lysozyme solution was decreased to ~1.5. The pH value and temperature of lysozyme solution after each addition of HCl stock were recorded.
Fluorescence titration. Fluorescence intensity measurements were performed at 25using an Aviv model ATF 105 spectofluorometer (Aviv Associates, Lakewood, NJ). The fluorescence intensity of lysozyme solution after each addition of HCl was measured by fluorescence spectroscopy. The titration profiles were measured by the incremental addition of aliquots of HCl to a 1cm path length cell containing 1.5 mL of lysozyme solution. Scans have been repeated three times to ensure the accuracy. The protein sample are exited at 295nm, the emission intensity were collected at 345nm through a monochoromator. For both pH titration and fluorescence titration, two trials were performed. The second trial was used to fill the gap between pH values and to make a smooth and accurate plot of pH versus intensity.
Circular dichroism. The CD spectra were recorded at 25 using an AVIV model 60 DS spectrophlarimeter (Aviv Associates, Lakewood, NJ). CD titration profiles were measured by incrementally adding HCl stocks each time to an optical cell containing 0.4mL of lysozyme. A 1mm cell was used for the CD measurements. The ellipticity was collected through wavelength of 210nm to 260nm.
Result
Figure 1. The urea density at different molar concentration.
As is shown above, the urea density increases with the growth of the molar concentration. Thus, we can use the linear as a calibration curve to calculate the density at a desired urea molar concentration.
Figure 2. Fluorescence spectra of lysozyme with 5M urea at acidic pH from 7.61-1.90.
Figure 2 tells that the fluorescence intensity of lysozyme presents a red shift at pH 2.93. Combined with Figure 1, the protein exhibits a maximum emission () wavelength of around 345 nm at acidic pH around 2.93. The shifts to around 358nm at pH 2.93 and begins to decline. The fluorescence intensity of lysozyme decreases gradually from ~16200 to ~14500 before pH reaches 2.93, while the intensity increases to ~18000 and the wavelength slightly shifts to right at pH 2.93. When the pH further decreases, the intensity significantly increases to around 29200, and the wavelength reaches around 358 nm. Then, from the graph above, we can see that the intensity gradually decreases again to 23600 as lysozyme titrated with more HCl buffer.
Figure 3. Fluorescence spectra of lysozyme with 3M urea at acidic pH from 7.07-1.67.
Likewise, Figure 3 shows that a relatively small red-shift occurred at acidic pH above 2.59. Lysozyme remains the same of around 345 nm before the pH decreased to 2.59, but an increase in was observed at acidic pH 2.59 or lower pH. Compared with Figure 2, the lysozyme with 3M urea could be seen to exhibit a smaller red shift in fluorescence intensity at a lower pH related to lysozyme with 5M urea. These two figures are examples that lysozyme at higher urea concentrations has a larger red shift at higher pH environment. The red shift appears at 3M or higher urea concentration, while lysozyme with urea concentration below 3M does not exhibit a shift in fluorescence intensity. The change in fluorescence maximum emission wavelength indicates a change in lysozyme structure, which suggests that no protein structure change at lower urea concentration even with the inducing of acidic pH. In addition, with the increase of the urea concentration from 0M to 8M, it becomes more difficult to decline pH of lysozyme solution down to around 1.5.
Figure 4. The lysozyme maximum intensity wavelength against aqueous environmental pH.
Figure 4 suggests that the wavelength increased with the decrease of environmental pH. The in the unfolded condition ranges from 340 to 345, while the shifts to 355-359 at low pH with the increase of urea concentration.
Figure 5. CD spectra of lysozyme solution with 3M urea.
Figure 5 shows that at pH 7.2, lysozyme exhibits a significant strucutre and there is a slight transition around pH 2.5. Also, upon a decrease in pH, the molar ellipticity only increases slightly, indicating that lysozyme was not fully denatured. Part of lysozyme may retain in structure.
Figure 6. CD spectra of lysozyme solution at 8M urea.
Figure 6 presents clear evidence for lysozyme unfolding. It can be seen that lysozyme retains before pH reaches around 3.5. The CD spectra at pH 2.24 is consistent with a random coil structure of lysozyme, but the molar ellipticity changes from -120000 to around -50000 and the change of relatively larger protein structure came into being at 8M urea than 3M urea.
Figure 7. Molar ellipticity of lysozyme with urea concentration from 0M to 8M at acidic pH 1.25-8.22.
Figure 7 shows the CD spectra of lysozyme at acidic pH of urea concentration from 0-8M. It tells that the molar ellipticity curves almost overlap above pH 3.5 without the effect of lysozyme concentration. Below pH 3.5, the lysozyme with 8M urea buffer has a pH and urea induced transition and the molar ellipticity gradually decreases with the decrease of urea concentration. Just slight or almost no transition could be seen in the lysozyme solutions of 0, 1 and 2 M urea buffer. But it could be seen that the transition midpoint shifts toward higher pH as the concentration of urea increases.
Figure 8. Molar ellipticity of lysozyme at 2 M urea concentration at 221 nm.
Figure 9. Molar ellipticity of lysozyme at 8M urea concentration at 221 nm.
Figure 9 tells that the molar ellipticity at 221 nm shifts slightly towards higher pH as temperature increases. Following the sigmoidal profile, the post denaturation baseline presents the completion of lysozyme denaturation and the lower baseline indicates that lysozyme retains in folded state above pH 3.5. Besides, a slight shift could be found in transition midpoint toward higher pH as the temperature increases.
Figure 10. Phase diagram of lysozyme denaturation. Midpoint transition pH is plotted as a function of urea concentration and temperature.
Figure 10 presents that the pH transition midpoint of lysozyme at 45 is generally higher than that at lower temperature. The blue linear is the steepest among all the three lines, while lines of lysozyme denaturation at 35 and 25 overlaps.
Discussion
Fluorescence intensity analysis. Figure 2 shows an increase in fluorescence intensity of lysozyme at 5M urea concentration around pH 2.93. This red shift may be attributed to a change in lysozyme structure. More specifically, the microenvironment of tryptophan (Trp) residues in lysozyme may change due to the cosolvent-protein interaction or the acid-induced denaturation. Exposure of a buried Trp to solvent results in a red shift of . The Trp residues in folded lysozyme present the maximum emission around 345 nm and the shift in to around 355 nm indicates the exposure of aromatic ring in Trp residue to aqueous environmental.^11 As for Figure, it tells that of lysozyme at 3M urea concentration has a smaller red-shift at pH around 2.59. Comparing Figure 2 with Figure 3, we can conclude that urea concentration plays an important role on lysozyme denaturation. As urea concentration increases, we can see a larger red shift and higher fluorescence intensity. In Figure 4, is plotted as a function of pH and urea concentration. When urea concentration increases, shifts to higher wavelength. Thus, urea concentration and lysozyme stability is found to have a close relationship, because urea arouses the denaturation of lysozyme by perturbing the intramolecular hydrogen bonding and the hydrophobic interaction between lysozyme molecules.^11 Also, it is found that higher urea concentration can lead to lysozyme less stable and easier to unfold.^12 Moreover, fluorescence intensity can become higher with the appearance of the transition, indicating a greater extent of unfolded lysozyme.
CD spectra analysis. Through the measurement of fluorescence intensity, lysozyme is found to undergo a conformational transition around pH 2.5-3. Our CD spectra provide more details about the change of protein secondary structure influenced by the urea concentration and acidic pH environment. Different structure will generate distinct CD spectra, so a small shift in molar ellipticity curve in Figure 6 tells us that lysozyme with 3 M urea concentration will have small conformational changes under the acidic conditions. And we can say that most of the lysozyme at 3 M urea retains some α-helix structure. Figure 7, meanwhile, presents a clear transition between α-helix and random coil. Therefore, a conclusion is drawn that lysozyme at higher urea concentration shifts to unfolded state more than that at lower uran concentration, witnessed from both Figure 6 and Figure 7.
Besides, from Figure 8 and Figure 9 above, we can find that temperature has an influence on protein unfolding: as temperature increases, the conformational transition of lysozyme comes into being at higher pH and lower urea concentration. At 45℃, the conformational transition of lysozyme at 8 M urea concentration occurs at pH around 4. Likewise, at the same temperature, part of lysozyme at 2 M urea concentration undergoes structure transition and the other part retains in strucutre. These two figures suggest that lysozyme is less stable and easier to change its conformation at higher temperature, whose reason is that higher temperature makes protein structure less stable. On the other hand, when temperature increases, the free energy required to unfold lysozyme decreases. Lysozyme retains its folded state, but it becomes less stable and is easier to overcome the intramolecular interaction between hydrophobic groups in lysozyme. Therefore, lysozyme is easier to unfold under the influence of urea and pH environment. And no structural transition occurs over the urea concentration range 0 to 2M except for lysozyme at 45.
Phase diagram analysis. As is shown in Figure 10, there are phase boundaries between lysozyme folding and unfolding phases. Area above or below boundaries is defined as the native state or the denatured state respectively. All three boundaries in the phase diagram of lysozyme have the positive slope, that is, the higher the urea concentration and the midpoint transition pH are, the more easily a conformational transition can occur. In addition, lower pH is required to denature 50 % of lysozyme at 25℃ and 35℃.
During the first stage of study, we have collected the fluorescence intensity and CD spectra at 0-8M urea concentration and constructed the phase diagram of lysozyme denaturation. Based on the results, subsequent experiments, including volumetric measurements of lysozyme structural transition, will be carried out in the second stage of study to further evaluate lysozyme structural transition.
Conclusion
By measuring the fluorescence intensity of lysozyme at urea concentration at range from 0 to 8 M, we suggest that shifts toward higher wavelength at pH ranging from 2 to 3 over the range of urea concentrations 3 to 8 M. As the urea concentration increases, will have a larger shift at lower pH. Meanwhile, through recording CD spectra, we ensure that lysozyme undergoes different structural alterations during the experiment, which can tell the secondary structure of lysozyme under the effect of pH and the presence of urea at three different temperatures. With our efforts of experiment and observations, we find that the results of CD measurements confirm to the fluorescence measurements and no structural transition occurs over the urea concentration range 0 to 2M except for lysozyme at 45. Lysozyme becomes less stable and easier to undergo a conformational change at higher temperature. Thus, the phase diagram shows lysozyme native/denatured state at different conditions, which better represents the relationship between protein stability and pH, urea concentration and temperature.
References
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