Mechanical Insight into Resistance of Betaine to Urea-Induced Protein

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Mechanical Insight into Resistance of Betaine to Urea-Induced Protein Denaturation Jiantao Chen,† Xiangjun Gong,*,† Chaoxi Zeng,‡ Yonghua Wang,‡ and Guangzhao Zhang*,† †

Faculty of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, P. R. China College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, P. R. China

‡

S Supporting Information *

ABSTRACT: It is known that urea can induce protein denaturation that can be inhibited by osmolytes. Yet, experimental explorations on this mechanism at the molecular level are still lacking. We have investigated the resistance of betaine to the urea-induced denaturation of lysozyme in aqueous solutions using low-field NMR. Our study demonstrates that urea molecules directly interact with lysozyme, leading to denaturation. However, betaine molecules interacting with urea more strongly than lysozyme can pull the bound urea molecules from lysozyme so that the protein is protected from denaturation. The number of urea molecules bound to a betaine molecule is given under different conditions. Proton NMR spectroscopy (1H-NMR) and Fourier transform infrared spectroscopy reveal that the interaction between betaine and urea is through hydrogen bonding.

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INTRODUCTION As an effective protein denaturant, urea has long been used in in vitro protein unfolding.1,2 The mechanism of urea-induced protein denaturation has been extensively studied.3,4 Generally, two mechanisms have been proposed. In the so-called indirect mechanism, urea is thought to induce protein denaturation by disturbing water hydration networks.5 Alternatively, urea molecules are suggested to increase the solubility of hydrophobic components by directly interacting with protein in a direct mechanism.6,7 The latter is supported by some recent simulations and experiments.8−12 Actually, urea is widely distributed in various organisms consisting of proteins. Yet, it never influences the work of urearich biological tissues. This is because some organic molecules acting as osmolytes in the organisms can effectively counteract protein denaturation and maintain the function of tissues.13 Betaine is one of the common osmolytes existing in plants and animals.14 It effectively inhibits urea-induced protein denaturation in mammalian kidneys.15 Near-infrared spectroscopy studies demonstrate that betaine as a strong water-structure former is excluded from protein and stabilizes the hydration shell of native proteins, which is called the exclusion mechanism.16 Vapor pressure osmometry studies show that betaine has unfavorable interactions with the hydrated domain surrounding the proteins,17−19 so it can prevent proteins from unfolding by reducing their exposed surface. The exclusion mechanism is supported by molecular dynamics simulation.20,21 However, recent simulations showed that the binding of betaine and urea is related to hydrogen bonds and van der Waals interactions should not be neglected.4,22 Another © XXXX American Chemical Society

simulation study further revealed that betaine was considered to form a complex with urea and water molecules, thereby pulling urea from the surface of the protein.23,24 Despite the above achievements in the resistance of osmolytes to protein denaturation, rare experimental studies quantitatively explore how osmolytes interact with urea, water, and protein molecules.25−27 Besides, although we previously studied the deep freezing-point depression in a urea−betaine mixture,28 the origin of the interactions between urea and betaine has not been explored experimentally yet. NMR spectroscopy is a powerful tool to study molecular diffusion and interactions.29 Particularly, low-field NMR (LFNMR) gives the spin−spin relaxation time (T2) and its distribution providing information about molecular mobility in either the liquid or solid phase.30−32 In the present study, we have investigated the interactions existing in a mixture of lysozyme, urea, and betaine in aqueous solution using LF-NMR equipped with an inverse Laplace transform spectrum.33 We measure and compare the interactions between lysozyme, water, urea, and betaine molecules. We reveal that the direct interactions between urea and lysozyme are the reason for the denaturation, whereas the interactions between urea and betaine prevent denaturation. Quantitative description of the interactions between urea and betaine, namely, the number of urea molecules bound by one betaine molecule, is given. Finally, proton NMR spectroscopy (1H-NMR) and Fourier Received: October 7, 2016 Revised: November 5, 2016 Published: November 7, 2016 A

DOI: 10.1021/acs.jpcb.6b10172 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B 1 − Pb P 1 = + b T2 T2f T2b

transform infrared (FTIR) spectroscopy show that these interactions result from the hydrogen bonding.

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(3)

where Pb is the percentage of bound water. On the other hand, molecules containing −OH, −COOH, −NH2, and −SH groups can chemically exchange protons with each other. This chemical exchange is diffusion-controlled.38 It happens only when the unlike molecules encounter with enough time.39 Equation 3 is also applicable to systems with a chemical exchange of protons.40 Proton NMR Spectroscopy (1H-NMR) Measurements. 1 H-NMR spectrum was recorded on a Bruker AV600 NMR spectrometer using D2O as the solvent at 25 °C. FTIR Spectroscopy Measurements. FTIR spectra were recorded on an FTIR spectrometer (Bruker VERTEX 70) over the energy range of 4000−450 cm−1 at 25 °C. Samples were prepared by dissolving urea, betaine, and lysozyme in D2O. Here, we used D2O instead of H2O because the bending mode of D2O locates at 1200 cm−1, which does not overlap with amide I band.

EXPERIMENTAL SECTION Materials. Lysozyme (Mw = 14 388, lyophilized powder) from chicken egg white with an activity of above 23 500.0 U/ mg was obtained from Calbiochem. Urea from Aladdin (≥99.5%), betaine from Admas-beta (≥98%), and poly(ethylene glycol) (PEG, Mn = 2000 g/mol) from Aladdin were used as received. Aqueous solutions of urea with concentrations ranging from 0.5 to 8.0 M were prepared using deionized (DI) water (Millipore, resistivity = 18.2 MΩ cm) and were stirred overnight at room temperature. Aqueous solutions containing urea and betaine (UB solution) were prepared with urea concentrations (Cu) ranging from 0.5 to 8.0 M and a betaine concentration (Cb) of 2.0 M. In addition, lysozyme solutions were prepared by dissolving lysozyme in DI water, urea solutions, betaine solutions, and UB solutions with a lysozyme concentration (Cl) of 1.4 × 10−3 M. An aqueous solution of urea (4.0 M) with PEG (5.6 × 10−3 M) was also prepared for comparison. LF-NMR Measurements. A Bruker mq20 bench-top NMR spectrometer operating at 20 MHz was used to obtain a magnetization decay curve. Each sample was inserted in a probe with φ = 10 mm, and the volume of each sample was measured to be around 0.5 mL. The Carr−Purcell−Meiboom−Gill pulse sequence was used to measure the spin−spin relaxation time (T2).34,35 Typically, 8000 data points were collected for each scan with a scan number of 8. The spacing between 90° and 180° pulses was 1 ms, and a recycling delay between the scans of at least 5T1 was used to ensure full recovery of the magnetization between acquisitions. The relaxation measurements were performed at 25 °C. The exponential fit is a common approach used to obtain T2 from the relaxation curve

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RESULTS AND DISCUSSION Urea−Lysozyme (UL) Aqueous Solutions. Normalized relaxation decay curves for pure water, urea solution, UL solution, and urea−betaine−lysozyme (UBL) solution are shown in Figure 1a. Inverse Laplace transform is used for the extraction of the distribution of the relaxation time (T2) for

⎛ t ⎞ ⎛ t ⎞ My(t ) = My1(0) exp⎜ − 1 ⎟ + My2(0) exp⎜ − 2 ⎟ + ··· ⎝ T22 ⎠ ⎝ T21 ⎠ (1)

where My(0) is the transverse magnetization immediately after the 90° pulse.36 As a result, the number and value of T2 is based on the number of exponential decays chosen. Instead, in our cases, another approach based on the inverse Laplace transform algorithm, CONTIN, was used to extract the spin−spin relaxation time (T2) inversion spectrum of multicomponent systems t ⎞⎟ ⎟ ⎝ T2j ⎠ ⎛

M (t ) =

∑ Ij exp⎜⎜− j

(2)

where Ij is the signal intensity of component j, whose spin−spin relaxation time is T2j. The location of T2j distribution is indicated by the optimal relaxation time (T2p). The peak area is defined as the sum of intensity at all points. T2p and the peak area were determined using the inversion software (Bruker). In the fast exchange model,37 water molecules adsorbed on a macromolecule or particles would exchange with those freely moving in the bulk and T2 is the weight average of the relaxation time for protons of bound water (T2b) and free water (T2f), where T2b < T2f. Thus, T2 is written as

Figure 1. (a) Relaxation decay curves of pure water (○), lysozyme solution (Δ), urea solution (Cu = 4.0 M) (×), and UL solution (Cu = 4.0 M) (●). The solid lines are the results fitted by the singleexponential (for pure water, lysozyme solution, and UL solution) and double-exponential (for urea solution) functions. The reduced chisquare values are 0.027, 0.017, 0.034, and 0.035, respectively. (b) Relaxation time (T2) inversion spectrum for pure water (a), lysozyme solution (b), urea solution (Cu = 4.0 M) (c), and UL solution (Cu = 4.0 M) (d). B

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The Journal of Physical Chemistry B each component. First, to confirm the reliability of this approach, we compare the relaxation time inversion spectrum and the exponential fit that are commonly used (see Figure S1 of the Supporting Information). It is shown that the center values of T2 from the two components are almost the same for both approaches. Figure 1b shows the relaxation time (T2) inversion spectrum for urea, lysozyme, UL aqueous solutions, and pure water, where the urea concentration (Cu) is 4.0 M, which is high enough to induce lysozyme denaturation.41 For pure water, only a unimodal distribution (T2p = 2.360 s) is observed as expected. The lysozyme solution also exhibits one peak (T2p = 2.313 s) close to that due to the protons of pure water. In principle, some water molecules are bound on the lysozyme surface and they coexist with the free water molecules. Due to the fast exchange, the T2 of water in solution is contributed by the T2 of bound water and free water based on eq 3. Moreover, the contribution of T2 of bound water to that of the water in solution causes the decrease in T2 compared with that of pure water. However, we did not observe any signal indicating the protons of lysozyme. This is because lysozyme has a compact structure, where the T2 of the protons is too small to be detected by LF-NMR.42 For the urea solution, we observe two peaks. The peak with a higher T2 (T2p = 1.732 s) can be attributed to the protons of water as it is close to that of pure water. However, the former is lower than the latter (T2p = 2.360 s), indicating that the water molecules move more slowly in urea solution. It is reported that the T2 of the protons of water molecules decreases in glucose solution because their motion is restricted due to the formation of hydrogen bonds between water and glucose.43 Likewise, water and urea also form hydrogen bonds, which restrict the motion of water molecules. It is important to note that urea with −NH2 cannot chemically exchange protons with water here because the hydrogen bonds between them have a short lifetime or their encounter is not long enough.44 The peak with lower T2 (T2p = 0.044 s) is attributed to the protons of urea. It is even much lower than that of protons of the restricted water molecules. This is because the quadrupole moment of 14N in urea decreases the relaxation time.40 Figure 1 also shows that the UL solution has only one peak at T2p = 0.202 s. Namely, the presence of lysozyme induces the chemical proton exchange between urea and water. To further understand this phenomenon, we examined the urea concentration dependence of relaxation time for urea solution and UL solution. Figure 2 shows T2p in urea solution or UL solution as a function of Cu. As Cu increases, the T2p of the protons of water in urea solutions gradually decreases, whereas that of the protons in urea slightly varies. It is known that the dipole− dipole interaction between urea molecules can decrease their mobility, leading to the decrease in T2. However, the coupling between the quadrupole moment of 14N and the local electric field gradient of 1H in urea molecules dominates the relaxation of protons, so that the effect of the dipole−dipole interaction can be neglected.36 Yet, T2p for the UL solution sharply decreases with increasing Cu. Assuming that urea molecules spontaneously bind to lysozyme molecules, the urea concentration dependence of T2 for UL solutions can be described by eq 3, where Ps is defined as the percentage of protons of urea in all protons in the solution detected. As discussed above, no chemical exchange of protons happens between free urea and free water molecules in urea solution because of their short encounter. However, once

Figure 2. Urea concentration (Cu) dependence of relaxation time (T2p) for protons of urea (○) and water (□) in urea solution and for protons of urea and water (Δ) in UL solution. The dashed line shows the T2 of the protons in the UL solution fit by eq 3.

urea molecules bind on the lysozyme surface in UL solution, they have enough time to exchange protons with the water molecules bound there. Meanwhile, the bound water molecules have a fast exchange with the free water molecules causing their T2 to merge. As a result, the protons detected exhibit the same T2. This is why we observe only one peak. This hypothesis is supported by the comparison between the area of a single peak in UL solution and the sum of the area of peaks in urea solution (see Figure S2). As their values are almost the same, we confirm that the single peak in UL solution is contributed by the protons of urea and water. To further confirm that the peak in the UL solution is the result of the chemical exchange of protons and fast exchange of molecules, we replaced lysozyme with PEG in the solution, where PEG does not interact with urea.45 We can observe three peaks in the spectrum, which are attributed to the protons of urea, PEG, and water (see Figure S3). As in the case of the urea solution, the peaks attributed to the protons of urea and water do not merge in the presence of PEG. Accordingly, chemical proton exchange does not occur between the free urea and bound water in UL solution. Instead, it occurs between the bound urea and bound water, where the latter acts as the intermediate between urea and free water. It is important to note that although urea molecules bind to lysozyme, some of the bound water molecules are displaced, which destroys the hydration shell of the protein. This initiates protein denaturation. Thus, our study supports the direct interaction mechanism of urea-induced protein denaturation.6 UBL Aqueous Solutions. Now, we turn our attention to the resistance of betaine to the urea-induced denaturation. First, we examined betaine and betaine−lysozyme (BL) solutions. Figure 3 shows only one peak in the spectrum for either the betaine or BL solutions. Furthermore, they exhibit similar relaxation times. Namely, T2p is 1.203 s for the former and 1.104 s for the latter. It is known that the protons of −CH2 and −CH3 in betaine are unexchangeable,46 that is, they cannot chemically exchange with those of water. However, as betaine has a low molecular weight, it has a mobility in solution similar to water. Thus, the protons of betaine and water in betaine solution have similar relaxation times and exhibit one peak. Figure 4 shows the betaine concentration (Cb) dependence of relaxation time (T2p) for the betaine solution or BL solution. Clearly, almost the same dependence is observed in the two solutions. Namely, the presence of lysozyme has little impact on the mobility of betaine. Accordingly, the interaction between C

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in the solution. For the UBL solution, we can observe three peaks at T2p = 0.013, 0.240, and 1.050 s, which are attributed to the protons of urea bound to betaine, protons of urea−water with chemical exchange and fast exchange, and the protons of betaine−water, respectively. Clearly, they shift so much relative to those in the UB solution without lysozyme because of the chemical proton exchange between urea and water, as well as the fast exchange between bound water and free water in the presence of lysozyme. Figure 6a shows that the T2p of the protons of betaine and water decreases as Cu increases, indicating that betaine and

Figure 3. Relaxation time (T2) inversion spectrum for the betaine solution (Cb = 2.0 M) (a) and BL solution (Cb = 2.0 M) (b).

Figure 4. Betaine concentration (Cb) dependence of relaxation time (T2p) for betaine solution (○) and BL solution (Δ).

betaine and lysozyme is very weak and it cannot be detected using LF-NMR. This is consistent with the results reported before, where betaine was found to be excluded from the protein surface.47 Figure 4 also shows that T2p decreases with Cb, indicating that the mobility of betaine and water decreases due to the restriction via hydrogen bonding.21 Figure 5 shows the relaxation time (T2) inversion spectrum for urea−betaine (UB) and UBL solutions. Like the urea solution, the UB solution exhibits bimodal distribution. The peak at T2p = 0.034 s is attributed to the protons of urea as it is quite close to that for the urea solution (T2p = 0.044 s). The other peak (T2p = 0.817 s) is attributed to the protons of water

Figure 6. Urea concentration (Cu) dependence of the relaxation time (T2p) of the protons of (a) betaine−water (○) and urea (Δ) in UB solutions and (b) urea−water (∇) and betaine−water (□) in UBL solutions. The dashed curve shows this dependence for urea−water in the UBL solution fit by eq 3.

water molecules move more slowly because of the increase in the interactions of urea with betaine and water. Meanwhile, the T2p of the protons of urea is similar to that in urea solution due to the quadrupole moment of 14N. As there are less water molecules in the UBL solution than in the UL solution at the same Cu, the percentage of protons of urea in the former is higher than that in the latter. Assuming that all urea molecules in the UBL solution are bound to lysozyme surfaces, we can predict T2 using eq 3. Figure 6b shows that the theoretical value of T2 regarding the protons of urea−water is lower than that measured using LF-NMR. In other words, only parts of the urea molecules are bound to lysozyme and are involved in the chemical proton exchange with the bound water. Defining Cu′ as the effective urea concentration or the concentration of urea molecules directly interacting with lysozyme, we can estimate Cu′ by analyzing T2p and peak area in terms of eq 3 (see the Supporting Information). Corresponding to the urea concentration for protein denaturation Cu = 4.0−8.0 M,39 Cu′ is 2.9−5.8 M in the presence of betaine. This shows that betaine can effectively reduce the urea molecules directly interacting with the protein.

Figure 5. Relaxation time (T2) inversion spectrum for the UB solution (Cu = 4.0 M, Cb = 2.0 M) (a) and UBL solution (Cu = 4.0 M, Cb = 2.0 M) (b). D

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The Journal of Physical Chemistry B This is why the addition of betaine can prevent urea-induced denaturation. Defining Nlu as the number of urea molecules bound to one lysozyme molecule and Nbu as the number of urea molecules bound to one betaine molecule, we have Nlu from Cu′ and calculate Nbu in terms of (Cu − Cu′)/Cb. Clearly, one lysozyme molecule is covered by thousands of urea molecules (Nlu = 2077−4109 for Cu = 4.0−8.0 M), leading to the unfolding of the protein. On the other hand, Figure 7 shows that Nbu

Figure 8. 1H-NMR spectra of urea, UB, UL, and UBL in D2O.

Figure 7. Number (Nbu) of urea molecules bound to one betaine molecule in the dependence of Cu in the UBL solutions (Cb = 2.0 M).

increases from 0.2 to 1.2 as Cu increases from 0.5 to 8.0 M. It indicates that the counteraction effect of betaine is stronger at a higher urea concentration. One betaine may bind with more than one urea molecule in the solution with a higher urea concentration. The reason for this may be that betaine has more opportunities to bind with urea in a urea-rich environment. It is important to note that we added urea solution into BL solution in the experiments discussed above. We also examined UBL solution with the addition of betaine solution (Cb = 4.0 M) into UL solution (Cu = 8.0 M), where lysozyme was already denatured (see Figure S4). We found that this makes no difference in the relaxation time (T2) inversion spectrum. Namely, betaine can pull the bound urea molecules away from the lysozymes. However, some urea molecules can never leave the lysozyme in the presence of betaine due to the equilibrium. Thus, urea interacts with either protein or betaine, but the latter dominates over the former. To understand the nature of the interactions, we examined the solutions using 1H-NMR and FTIR. Figure 8 shows the 1H-NMR spectra for urea, UB, UL, and UBL in D2O. The signal at 5.75 ppm in the spectrum of urea is attributed to −NH2. The addition of lysozyme to urea solution does not vary the chemical shift for urea. Besides, the chemical shift for urea in the spectrum of UBL is the same as that in the UB solution. It was deemed that urea and lysozyme form hydrogen bonds or electrostatic interactions.3,48 The 1H-NMR results here demonstrate that they do not interact via hydrogen bonding, so the electrostatic interactions should be responsible for the protein denaturation. On the other hand, the addition of betaine to the urea solution leads to the chemical shift of urea at 5.82 ppm, further indicating the formation of hydrogen bonds between urea and betaine. Figure 9 shows FTIR spectra of UL, BL, and UBL in D2O. The band attributed to νasNH2 at 3435 cm−1 in the UL−D2O solution shifts to 3428 cm−1 and becomes broader after betaine

Figure 9. FTIR spectra of UL, BL, and UBL in D2O.

is introduced. This is because urea and betaine form hydrogen bonds (N−H···N and N−H···O).49 This hydrogen bonding leads to the association of −NH2, inducing a downward shift of νasNH2. Likewise, betaine and urea were reported to form hydrogen bonds in a deep eutectic mixture.28 Anyhow, the FTIR and 1H-NMR measurements confirm the hydrogen bonding between betaine and urea. Clearly, it can overcome the electrostatic interactions between urea and lysozyme, so that betaine can pull urea away from the surface of the lysozyme. This is why betaine can protect proteins from denaturation.

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CONCLUSIONS We have investigated the spin−spin relaxation time of protons of urea, betaine, and lysozyme in aqueous solutions using LFNMR. The interactions between water, urea, betaine, and lysozyme can be well characterized by the relaxation time (T2). Chemical proton exchange happens between urea molecules and water molecules bound to the protein, which correlates with the fast exchange between the free and bound water molecules. The bound urea increases the solubility of protein chains and makes protein denaturation possible. On the other hand, we found that the addition of betaine before or after the lysozyme was denatured makes no difference in the relaxation time (T2) inversion spectrum. This indicates that betaine can protect the lysozyme by pulling urea from the surface of the protein. The number of urea molecules bound to a betaine molecule increases with the increase of urea concentration. Further measurements using 1H-NMR and FTIR show that betaine molecules form hydrogen bonds with urea molecules. This hydrogen bonding can overcome the interactions between urea and the protein, so that urea-induced protein denaturation is inhibited. E

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b10172. Relaxation time obtained by the inversion spectrum and the double-exponential fit; peak area for urea and water from the inversion spectrum in dependence of the concentration of urea in urea solution and UL solution; relaxation time inversion spectrum for urea−PEG aqueous solution; relaxation time inversion spectrum for UBL solution; calculation of effective concentration of urea (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.G.). *E-mail: [email protected] (G.Z.). ORCID

Xiangjun Gong: 0000-0001-7049-944X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the National Natural Science Foundation of China (21234003) and the Fundamental Research Funds for the Central Universities is acknowledged.

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REFERENCES

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DOI: 10.1021/acs.jpcb.6b10172 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jpcb.6b10172 J. Phys. Chem. B XXXX, XXX, XXX−XXX