Direct Anionic Effect on Water Structure and Indirect Anionic Effect on

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

pubs.acs.org/JPCB

Direct Anionic Effect on Water Structure and Indirect Anionic Effect on Peptide Backbone Hydration State Revealed by Thin-Layer Infrared Spectroscopy Juan Zhao* and Jianping Wang Beijing National Laboratory for Molecular Sciences; Molecular Reaction Dynamics Laboratory, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China University of Chinese Academy of Sciences, Beijing 100049, P. R. China ABSTRACT: In this work, the anionic effect on water structure and on the peptide backbone and water interaction was investigated directly in aqueous solution using thin-layer transmission infrared spectroscopy. The chaotropic anions were found to weaken the water hydrogen-bonding strength and red shift the HOH bending frequency, while the kosmotropic anions were found to strengthen the water hydrogen-bonding network and blue shift the HOH bending frequency. The kosmotropes, especially F−, blue shift the vibrational frequencies of both amide II and amide III bands of N-methylacetamide (NMA), indicating NMA is in the “salting-in” state; while the chaotropes (Cl−, NO3−, Br−, I−, and SCN−) red shift the frequencies of the two normal modes, indicating NMA is in the “salting-out” state. Furthermore, the changes of the vibrational frequencies of the HOH bending, amide II and III bands were found to generally follow the Hofmeister anionic series. Our results suggest that hydrated anion influences the peptide backbone mainly through the N−H group, but a weak and indirect effect through the amide CO group also contributes. Thus, these amide modes can be used as vibrational measures of anionic influences on peptide backbone’s hydration state. Our work also suggests that deuteration of the amide unit decreases the sensitivity of the amide II and III vibrational modes in this regard. An ultrafast infrared spectroscopic study17 showed that ion has no influence on the water’s hydrogen-bonding network outside the first solvation shells of the ion, except the high valence ion. However, the neutron diffraction experiment indicates that the ionic effect on the structural perturbation of water goes beyond the first hydration shell of the ion.16,18 Infrared spectroscopy is known to be sensitive to molecular structures and their distributions. In pure water, the symmetric and asymmetric O−H stretching modes (3100−3500 cm−1) are indistinguishable because of the hydrogen-bonding network.19 However, the infrared spectrum of the HOH bending vibration (peak position is located at ca. 1650 cm−1) is known to be narrow in spectral width and moderate in intensity among water vibrations,20 which provides an effective probe to study the ion effect on water structure. In particular, under a “thinlayer” condition, which is referred to as liquid samples with a thickness of a few micrometers in this work, the bending mode will have a reasonable optical density in its infrared absorption, unlike the case of the OH stretching mode that is easily to be saturated. Ultrafast water dynamics has been studied directly using a few-micron thick sample for pure water.20−22

1. INTRODUCTION The anion influences the hydrogen-bonding network and the activity of water in a direct way by forming anionic solvation shells,1−4 which is also the onset of indirect impact on bulk water. Further, anion may directly or indirectly influence the biophysical and biochemical processes in solution phase, such as protein folding, protein stability, and aggregation.5,6 The ionic effect on protein has been believed to follow the wellknown Hofmeister series, which are ranked by their abilities of affecting the solubility of proteins.7 The Hofmeister anionic series is known to be CO32− > SO42− > H2PO4− > F− > Cl− > Br− > NO3− > I− > SCN− > ClO4−, where the ions on the left side of chloride were referred to as kosmotropes, which strengthen water hydrogen bonding (HB) and lead to a greater salting-out degree of protein. The anions on the right side of chloride were referred to as chaotropes, which weaken water hydrogen bonding and lead to a greater salting-in degree of protein. However, the anionic effect on peptide backbone conformation, on the other hand, has never been proven to be a direct effect or an indirect effect yet. Recently, the ionic effect on water structure and the hydrogen-bond structural dynamics of water has been examined using the O−H or O−D stretching of HOD in H2O or D2O by linear and ultrafast nonlinear infrared spectroscopies.8−14 However, the spatial extent of the ion effects on the hydrogen-bond structure of water remains controversial.15,16 © XXXX American Chemical Society

Received: September 27, 2017 Revised: November 18, 2017 Published: December 12, 2017 A

DOI: 10.1021/acs.jpcb.7b09591 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

This work is organized as follows. The anionic effect on the water hydrogen-bond structure was first analyzed using the infrared spectrum of the HOH bending mode, using a series of saline solutions (Na2CO3, Na2SO4, NaF, NaCl, NaBr, NaI, NaNO3 and NaSCN) at M-level of concentrations. Then the peptide−anion interaction was examined using the infrared spectra of the amide I, II, and III bands directly in water, using the thin-layer infrared spectroscopy in which the HOH bending mode can be subtracted. For this purpose, nondeuterated Nmethylacetamide (NMA-h7, or simply NMA) that has one amide unit and two methyl groups nearby is employed so that the CO and N−H groups expose to solvent directly and to their maximum extent. Infrared spectral change of such water, peptide, and anion complexes was examined carefully in different saline solutions in water bending, amide I, II, and III vibrations region, from which the anionic effect on water structure and on peptide backbone in peptide/water system was examined.

For a given peptide, the peptide−water direct contact in saline solution resembles the salting-in state of the peptide, while a nonwater contact situation resembles the salting-out state of peptide. These states will definitely be sensed by the amide vibrations that are of both peptide conformation and solvent environment sensitivities. Amide group is the basic repeating component of peptides or proteins along the backbone. Up to nine amide vibrational normal-mode vibrations are known to be associated with an amide unit (CONH), which are sensitive to the microenvironment of peptide backbone, and also to their secondary and high-order structures. Among these normal modes, the amide I mode (1700−1600 cm−1), which is mainly the CO stretching vibration, is usually used as a conformation marker of peptides and proteins due to its large dipole moment.23−28 Recently, the cation−peptide interaction has been found to cause the infrared spectral change in the amide I band.29−31 These results suggested that the divalent cations (Ca2+ and Mg2+) may interact with the carbonyl group, while the monovalent cations may not. How anion influence the amide I mode has not been reported so far. The amide II and amide III modes, on the other hand, are mainly the combination of the C−N stretching and the N−H in-plane bending vibration (Scheme 1). The difference between

2. MATERIALS AND METHODS 2.1. Materials. N-Methylacetamide and all of the salts used in this work were purchased from J&K Chemical and used without further purification. The salts used here include Na2CO3, Na2SO4, NaF, NaCl, NaBr, NaI, NaNO3, and NaSCN. The concentration of salt in H2O is 1 M for Na2CO3, Na2SO4, and NaF and 2 M for the remaining salts. NMA was dissolved in these saline solutions at a concentration of 2 M. 2.2. Thin-Layer FTIR Spectroscopy. Infrared spectra of H2O in saline water and solvated NMA in various saline solutions were measured using a Nicolet 6700 FTIR spectrometer equipped with a liquid-nitrogen-cooled mercury−cadmium−telluride detector. The samples were placed in a homemade IR sample cell consisting of two 2 mm thick CaF2 IR-optical windows separated by a 6-μm thick Teflon spacer. In such a way, the HOH bending mode of pure water at ca. 1650 cm−1 shows an optical density of 0.8−0.9 OD, which allows one to examine the water/ion interaction in the HOH bending frequency region as well as peptide/water interaction in the amide I and amide II region (1700−1550 cm−1) under the influence of various anions. The spectral resolution of IR spectra was 1 cm−1, and each sample was averaged over 64 scans. Dry air was used to purge the FTIR spectrometer and its sample chamber during the spectral measurements. In saline solutions, the IR spectrum of H2O was subtracted from that of NMA to obtain the IR spectrum of NMA. To eliminate the effect of the HOH bending on the amide I and amide II bands, the subtracted spectrum in the 1750−2000 cm−1 region was kept relatively flat as suggested by a previous work during spectral subtraction.45 The peak position of a vibrational mode in an IR spectrum was determined by directly reading its peak position and also confirmed by the zero-intensity position of its first derivative. It should be mentioned that while the FTIR spectrometer used here has the spectral resolution set to 1 cm−1 only, the digital resolution can be as high as 0.1 cm−1. This digital resolution is relatively meaningful and helpful in describing difference FTIR spectra at various conditions. All of the IR measurements were carried out at room temperature (22 °C). 2.3. Molecular Dynamics Simulations. MD simulation of NMA in a simple single-atom ionic solution was performed using the NAMD program with CHARMM force field for NMA. The SPC/E model was used for water, which has been

Scheme 1. Water and Peptide Backbone Interactiona

a Water bending mode (δHOH) and peptide amide II mode (only the N−H in-plane bending motion) are shown.

the two modes is the amide II mode (1600−1500 cm−1) is an out-of-phase combination of the C−N stretching and the N−H in-plane bending, while the amide III mode (1350−1200 cm−1) is an in-phase combination of the C−N stretching and the N− H in-plane bending.32 These two vibrational modes are also sensitive to the environment and conformation of peptides and proteins and can be used as peptide structure probes.33−42 Our recent work43 showed that the amide II mode is sensitive to peptide backbone−cation interaction. Using fully deuterated Nmethylacetamide (NMA-d7), a recent work44 indicated that the vibrational frequencies of the amide I and amide II bands were not affected by anions even at very high concentrations. However, it is generally believed that anions do affect the solubility of proteins;5,14 thus, the anion and peptide backbone must have certain interactions, which will be manifested in these conformational marker bands. In addition, there is also an even lower-frequency amide vibration, namely the amide III band, that can be used to study the ion effect on backbone. These are the motivations of this work. B


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The Journal of Physical Chemistry B shown to be acceptable in describing the ionic effects on water structures as well as peptide conformations in aqueous solutions.15,16,46−49 The force-field parameters for Na, F, Cl, Br, and I were obtained from a previous work.49 Each MD ensemble contains 8 NMA molecules in a cubic solvent box with an initial size of 28 × 28 × 28 Å3, and the concentration of NMA is about 1 M. There are 8 pairs of ion in the solvent box with the salt concentration set to about 1 M. The nonbonded cutoff distance was set to 12 Å, and the particle mesh Ewald summation was used for long-range electrostatic interaction. Before the MD simulation, the equilibration run was performed to ensure a stable NPT ensemble. MD simulations (NPT ensemble) were finally performed using the Langevin-piston Nose−Hoover method for 1 ns with a step of 200 fs. The radial distribution function (RDF) of anion around water hydrogen and amide NH groups was obtained using the MD trajectories.

3. RESULTS AND DISCUSSION 3.1. Ionic Effects on the Hydrogen Bonding Structure of Water. We first analyzed the ionic effect on the water structure. In bulk water, one water molecule can statistically form a hydrogen bond with up to four water molecules around it. Such a clustered water structure is the origin of a broad distribution of the O−H stretching and the relatively narrow HOH bending vibrations observed in IR spectroscopy. The hydrogen-bond interaction weakens the O−H chemical bond, resulting in a decreased restoring force of the O−H stretching. Thus, increasing the hydrogen-bonding strength will decrease the frequency of the O−H stretching frequency. However, for the HOH bending mode (Scheme 1), the formed hydrogen bond tends to diminish the bending of the HOH that would alter the linear O−H···O hydrogen-bonding structure. Under such circumstances, the HOH bending force constant will increase. Thus, increasing hydrogen-bond strength will increase the HOH bending frequency.20 Our work here focuses on the bending of water. We first analyzed the concentration-dependent NaI effect on the water hydrogen-bonding network. The IR spectra of the HOH bending of H2O as a function of the NaI concentration are shown in Figure 1a. The peak position of the HOH bending mode in pure water is located at ca.1643.6 cm−1, with a slight asymmetric shape (strong at the high-frequency side and weak on the low-frequency side) that can be fitted with two Gaussian functions.20,50,51 However, as the salt concentration increases, the frequency of the HOH bending shows a progressive red shift, indicating a decreased water hydrogen-bonding strength. At 5 M NaI concentration, the peak position of the HOH bending is at 1626.5 cm−1 with a red shift of ca. 18 cm−1 from the case of pure water (1643.6 cm−1). Figure 1b shows the NaI concentration-dependent difference spectra. Here, an increased positive peak at the low-frequency side indicates an increased water−ion interaction, while an increased negative peak at the high-frequency side is due to the weakening of the original water−water interaction. Thus, the IR result presented here is in agreement with the Hofmeister anion effect7 that I− is chaotropes. In addition, as the salt concentration increases, the transition intensity of the HOH bending mode increases, agreeing with a previous Raman study of the HOH bending mode.52 The concentration effect of NaI on the water structure is similar to the temperature effect. As the temperature increases, the water molecules become weak hydrogen-bonded, leading to a red-shifted HOH bending frequency.53

Figure 1. Concentration-dependent IR spectra of the HOH bending mode of H2O in NaI solution (a) and their difference spectra with the case of pure water (b). Dashed line in panel b indicates the absorption peak position of the HOH bending mode in pure water.

With the above results established, we then examined other anionic effect on the water hydrogen-bonding network. Figure 2a,b shows the area-normalized IR spectra of the bending

Figure 2. Area-normalized thin-layer FTIR spectra of the HOH bending in kosmotropic (a) and chaotropic (b) anion solutions, respectively, and their corresponding IR difference spectra with respect to the case of pure water (c, d). The concentration of salt in H2O is 1 M for Na2CO3, Na2SO4, and NaF, and 2 M for the remainder salts. Dashed lines in panels c and d indicate the absorption peak position of the HOH bending mode in pure water.

vibration in pure water and in various sodium salt solutions, respectively. Anion-dependent peak positions are listed in Table 1. In the saline solutions, the ionic strength of anion is very similar to one another except for the case of F−. Figure 2a shows that in Na2CO3, Na2SO4, and NaF solution, the vibrational frequency of the HOH bending slightly increases, indicating an increased water hydrogen-bonding interaction. However, in other saline solutions (Figure 2b), the HOH vibrational frequency decreases, especially in NaI salt solution, indicating a decreased water hydrogen-bonding interaction. C


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Table 1. Anion-Dependent Vibrational Frequency (ν, in cm−1) of the HOH Bending (νHOH) of Water and Those of the Amide I, Amide II, and Amide III bands of NMA ωHOH ωAm−I ωAm−II ωAm−III

H2O

CO32−

SO42−

F−

Cl−

Br−

NO3−

I−

SCN−

1643.6 1625.2 1580.3 1315.6

1648.3 1625.6 1580.4 1315.4

1645.8 1625.4 1580.3 1315.6

1645.7 1625.2 1581.2 1316.0

1643.5 1625.3 1577.9 1313.7

1642.6 1624.1 1576.4 1312.6

1643.6 1625.4 1577.7

1640.6 1624.1 1574.5 1311.4

1642.2 1624.2 1576.5 1312.8

K−1 mol−1) was found to be between those of Cl− and I− (−36 J K−1 mol−1). Thus, the order of hydration entropy of these anions is consistent with their influences on water bending frequency shown in Scheme 2 but appears not to agree with the classical Hofmeister series listed on the top of Scheme 2. Most interestingly, except for the vibrational frequency of the HOH bending in saline solution, the peak-shape and bandwidth of the HOH bending mode are found to be different from the case of pure water. The peak width, in terms of full-width at half-maximum (fwhm), is generally narrower in saline solution than that in pure H2O, for both kosmotropic and chaotropic salts (except for NaF), suggesting a generally decreased structural inhomogeneity of water in the presence of anions. A previous work also showed that, as the salt concentration increases, the width of the Raman HOH bending mode frequency of water decreases.52 A linear relationship between the fwhm of the HOH bending and the frequency shift is shown in Figure 3, whose values and orders are also listed in

This is in agreement with the well-known Hofmeister anion series that CO32−, SO42−, and F− are kosmotropic anions, while Cl−, Br−, NO3−, I−, and SCN− are chaotropic anions. The difference spectra can be used effectively to reveal subtle structural change of a molecular system. Figure 2c,d shows the IR difference spectra of the HOH bending obtained by subtracting the infrared spectrum in pure water from those of saline solution. Positive peaks in the difference spectra are thus due to the ion effect. Positive peak higher than 1643.6 cm−1 indicates a blue shift of the HOH bending, while the positive peak lower than 1643.6 cm−1 indicates a red shift. It is clear in Na2CO3, Na2SO4, and NaF solution that the positive peak position is higher than 1643.6 cm−1. For other cases, the positive peak position is lower than 1643.6 cm−1. The results clearly confirm that CO32−, SO42−, and F− increase the strength of hydrogen bonding of water, while Cl−, Br−, I−, and SCN− decrease the strength of hydrogen bonding of water. The order of the frequency of the HOH bending is CO32− > SO42− ≈ F− > NO3− ≈ Cl− > Br− > SCN− > I−, a finding that is in reasonable agreement with a recent study of ions in water using the attenuated total reflection (ATR) IR method,54 where the anionic effect on the water structure was examined solely based on the HOH bending frequency. Based on these results, one sees that the change of the vibrational frequency of the HOH bending mode (Scheme 2) roughly follows the classical Scheme 2. Frequency Shifts (All in cm−1) of the HOH Bending of Water (ΔωHOH), Amide II (ΔωII) and III (ΔωIII) Modes of NMA in Various Saline Solutionsa

Figure 3. FHWM of the HOH bending as a function of its frequency shift in various saline solutions. The dash line indicates the fwhm of the HOH bending in pure water.

Scheme 2 (second row). The order of such change also roughly follows the Hofmeister anionic series (except for F− and NO3−) but not in the same sense of anion effect on water structure predicted by the series. In addition, it should be pointed out that some frequency shifts listed in Scheme 2 are insignificantly small (at the level of tenths of cm−1), which are obtained by directly reading its peak position and also confirmed by the zero-intensity position of its first derivative. The subtle difference between two peak positions can also be confirmed by taking difference spectra between two corresponding IR spectra. This suggests that these values are still relatively meaningful, even though these values are very close to the digital spectral resolution of the FTIR spectrometer used in this work. 3.2. Ionic Effect on Water−Peptide Backbone Interaction. The NaI concentration-dependent infrared spectra for the amide I, II, and III bands of NMA are shown in Figure 4a,b, while the corresponding difference spectra are shown in Figure 4c,d. In pure water, the peak frequencies of the amide I, II, and III bands are at 1625.2, 1580.3, and 1315.6 cm−1, respectively. A previous work56 reported that frequencies of the amide I, II,

HOH bending spectral width change (ΔFWHMHOH, in cm−1) was also listed. Pink blocks indicate abnormal Hofmeister order.

a

Hofmeister anion series. However, one also notes a few exceptions, which were found for polyatomic ions, such as NO3− and SCN−. The abnormal chaotropic effect of NO3− has been discussed in a previous work:54 the symmetric and extendable structure of NO3− may increase the regularity of the hydrogen-bond network of water and thereby tightens the water structure. In addition, the hydration entropy of NO3− (−76 J K−1 mol−1) was found to be close to that of Cl− (−75 J K−1 mol−1),55 hence the effect of NO3−on the water structure may be similar to Cl−. The hydration entropy of SCN− (−66 J D


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Figure 4. Concentration-dependent IR spectra of the amide I (Am-I), amide II (Am-II), and amide III (Am-III) bands of NMA in NaI salt solution (a, b) and their difference spectra with the case of pure water (c, d). Black dashed lines in panels c and d indicate the positions of the amide I, II, and III bands of NMA in pure H2O, respectively, while red dotted line can be regarded as the boundary of the amide I and II bands. Note: in H2O, peak intensity of these modes is roughly IAm−I/IAm−II/IAm−III = 1:0.5:0.1.

The NaI concentration-dependent infrared difference spectra of the amide I band in Figure 4c show an increased positive peak at the high-frequency side, indicating the CO group sees “less” amount of water at higher NaI concentrations. This is a very important finding because a previous work indicated that sodium does not bind to the CO group.30 Although the amide I band is mainly the CO stretching vibration (the contribution of the potential energy distribution of CO is ca. 80%),32,57 its vibrational frequency can be influenced by the hydrogen bond on the CO group directly and on the NH group indirectly. Our DFT calculation indicates the frequency of the amide I band red shifts ca. 10 cm−1, when an isolated NMA form a hydrogen bond with water on the NH side, but red shifts ca. 30 cm−1 when forming a hydrogen bond with C O group. In addition, the increased intensities of the positive peak in the difference spectra of the amide I, II, and III bands indicate that more NMA molecules are located in the “dehydration state” as the salt concentration increases. This also can be seen from the increased frequency difference between the positive and negative peak position for the amide I, II, and III bands (Figure 6). However, Figure 6 clearly shows that even though the amide II and III modes are sensitive to the anion concentration, the amide I appears to be more sensitive. Here the sensitivity is simply believed to be proportional to the frequency change as a function of ionic concentration. The reason will be discussed in Section 3.3. Based on the difference spectra of the amide III band, which is located in an undisturbed frequency region for NMA in H2O, we conclude that at 5 M NaI, about 22% NMA molecules locate in the dehydrated state, assuming an unchanged transition dipole of the amide III mode. The area-normalized FTIR spectra of the amide I, II, and III bands of NMA in the absence and presence of other different saline solutions are shown in Figures 7 and 8. The peak position results are also listed in Table 1 for comparison. It is clear that in Na2CO3 and Na2SO4 solutions, the vibrational frequency of the amide II band is almost unchanged. However, this band slightly blue shifts in NaF solution, which can be seen from the peak position listed in Table 1 and the positive peak between 1600 and 1580 cm−1 in the difference spectra, indicating the NMA is in the “salting-in” state in NaF. On the contrary, the main peak position of the amide II band and the positive peak position (higher than 1580.3 cm−1) in the difference spectra in chaotropic saline solutions show the amide

and III bands of NMA in gas phase are 1728, 1500, and 1259 cm−1, respectively. By comparing these frequencies to the observed solution-phase values listed in Table 1, one can see clearly that hydrogen-bonding interaction between peptide and water red shifts the amide I frequency and blue shifts the amide II and III frequencies. As the salt concentration increases, larger red-shifts in the vibrational frequencies of both amide II and amide III bands are seen, indicating a diminished weakened peptide−water interaction. In addition, the contribution of the N−H in-plane bending to the amide III band (ca. 30%) is smaller than that to the amide II band (ca. 40%), thus, the amide III band is less sensitive to the change of the hydrogen bond formed with amide NH group than the amide II band. Therefore, the frequency shift of the amide III band is smaller than the amide II band as the salt concentration increases, which can be seen clearly in Figure 5, where a comparison of

Figure 5. Relationship between the absolute frequency shift of the amide II and amide III bands as a function of the NaI concentration, with respect to the case of pure water. Dashed line indicates the diagonal line of the plot.

the frequency shifts of the amide II and amide III bands is given. Furthermore, the increased positive peaks in the difference spectra (Figure 4c,d) of the amide II and III bands at the low-frequency side indicate a reduced hydrogen-bonding interaction strength on the NH group, which is mainly caused by the ion hydration, especially the I− ion. More population of negative peaks is shown for both amide II and amide III bands, which is accordingly due to the weakening of the original peptide−water interaction. E


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Figure 8. Area-normalized FTIR spectra of the amide III bands of NMA in H2O and in kosmotropic (a) and chaotropic (b) saline solutions (1 M concentration for Na2CO3, Na2SO4, and NaF and 2 M for the remainder salts). (c, d) Corresponding difference spectra of the amide III bands obtained by subtracting the IR spectrum in pure H2O respectively. Black dashed lines in panels c and d indicate the peak position of the amide III band in pure water, respectively.

Figure 6. Absolute value of the frequency difference between the positive and negative peak positions in the difference spectra of the amide I (a), amide II (b), and amide III (c) bands, as a function of the NaI concentration. Dashed line in each panel is a linear fit to the data points.

NO3− vibration overlaps with the amide III band. The change of the infrared spectra of the amide III band in different saline solutions further confirms the results obtained in the amide II band. Similarly, in Na2CO3 and Na2SO4 solutions, the frequency of the amide III band still remains unchanged, but it slightly blue shifts in NaF solution. However, in chaotropic saline solutions, the frequency of the amide III band red shifts clearly. The frequency shift in NaI solution is also the largest, agreeing with the result in the amide II band. Furthermore, the characteristics of the spectral change of the amide II mode in the presence of F− and other chaotropic ions also can be more clearly seen in the difference spectra shown in Figure 8c,d, which particularly shows the opposite effect of the kosmotropes and chaotropes on the amide group. 3.3. Sensitivity of the Amide Modes on Anions. A recent report44 concluded that the vibrational frequencies of the amide I and amide II bands in deuterated NMA (NMA-d7) were not affected by anions even at high concentration condition. However, a careful examination of their data suggests limited frequency shift in the amide II mode, which can be understood by comparing the change of the nature of the amide II mode upon deuteration. Since there is a large contribution of the N−H in-plane bending to the amide II band, the vibrational frequency of the amide II band is significantly affected by the H/D exchange. The vibrational frequency of the amide II band in NMA-d7 decreases about 100 cm−1 from the case of NMA-h7. Potential energy distribution (PED)32 analysis presented in Table 2 shows that in NMA-d7 the amide II mode contains a significant amount of the C−N stretching (ca. 40%), while the contribution of the N−D bending to the amide II mode is only about 15%. Therefore, the hydrogen bonding on the ND site has a weak effect on the amide II band, and hence the infrared spectrum of the amide II band is less sensitive to the ion effect in NMA-d7. In other words, the anion does interact with amide NH group, but such interaction will not be shown in the IR spectra of deuterated NMA. Because the amide III mode is also a linear combination of the C−N stretching and N−H in-plane bending, we also listed the computational results of NMA-h7 and NMA-d7 in Table 2. The computed frequency of the amide III is assigned based a previous work.58 As can be

Figure 7. (a, b) Area-normalized FTIR spectra of the amide I and II bands of NMA (2 M) in H2O and in various saline solutions (1 M concentration for Na2CO3, Na2SO4, and NaF and 2 M for the remainder salts). (c, d) Difference spectra of the amide I and II bands obtained by subtracting the IR spectrum in pure H2O. Black dashed line in panels c and d indicates the peak position of the amide I and II bands in pure water, respectively. Red dot line can be regarded as the boundary of the amide I and II bands.

II frequency red shifts in these saline solutions, indicating the NMA is in the “salting-out” state. The largest red shift is about 5.8 cm−1, appearing in NaI solution. The main peak position of the amide I band slightly changes in various saline solutions compared with that in pure water, which can be seen from Figure 7a,b, and particularly in the difference spectra shown in Figure 7c,d, even though their peak positions appear to be within 1 cm−1 variation (Table 1). These frequency changes, although being small, are consistent with the change occurred in the amide II and III modes in all of the saline solutions evaluated in this work, demonstrating that these changes are real. In other words, the amide I mode is sensitive to the presence of anions. The area-normalized FTIR spectra of the amide III band in pure water and in various saline solutions are show in Figure 8a,b, and the peak position of the amide III band is also listed in Table 1. Here, NaNO3 solution is not considered, because F


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The Journal of Physical Chemistry B Table 2. Computed Vibrational Frequencies (ω, in cm−1) and PEDs of the Amide I, Amide II, and Amide III Bands in NMA-h7 and NMA-d7 mode

ω

PEDa

NMA-h7

amide I amide II amide III

1743.7 1559.8 1265.9

NMA-d7

amide I amide II

1732.8 1433.1

amide III

936.4

COs (81%), CCNb (5%) NHb (40%), HCHb (24%), CNs (17%) CNs (30%), NHb (29%), HCCNτ (10%), OCCb (8%) COs (85%), CCNb (5%) CNs (50%), NDb (15%), NCs (10%), CCs (8%), OCCb (7%) NDb (30%), NCs (24%), CCs (8%), CNs (6%), COs (5%), CNCb (6%)

the amide II and amide III bands remain nearly unchanged, but the HOH bending blue shifts in these two saline solutions. This suggests that CO32− and SO42− have insignificant effect on the water−peptide interactions. However, because these two anions do affect water bending frequency, this implies that they have no influence on the water’s hydrogen-bonding network outside their first solvation shell. Overall the data included in Figure 6 showed that the difference between the positive and negative peak positions of the amide II and III bands are not as significant as that of the amide I mode, as the NaI concentration changes. This can be explained as follows. We studied cationic effect on the amide I previously for a single peptide compound.29 It was found that peptide amide I mode frequency is cation concentration dependent. For sodium ion, the more pronounced spectral feature is the broadening of the amide I mode. This is also seen in the amide I spectral profile shown in Figure 4a,c. Thus, the extracted peak shift for the amide I shown in Figure 6 actually contains two contributions, one is from amide I as a consequence of Na+ effect, which could be the quite likely a major contribution, and the other is due to indirect influence of anion via the amide N−H group. Because the frequency shift of the amide I mode and that of the amide II mode is always in opposite direction, as shown in Figure 4, which is also wellknown for peptide (an extreme comparison is between gasphase frequencies and aqueous-phase frequencies for the amide I and II modes56). Under such circumstances, a blue shift of the amide I mode occurs when a red shift of the amide II mode occurs because of the NH and hydrated anion interaction. Unfortunately one cannot easily isolate these two contributions to the amide I mode from one to another. This explains the observed largest frequency shift of the amide I mode, as summarized in Figure 6.

s = stretching vibration; b = bending vibration; τ = torsional vibration. Only the contributions ≥5% are listed.

a

seen, the PED changes for the CNs (30% in h7 to 6% in d7) but not for the NHb to NDb. This suggests different sensitivities for the amide II and III modes to peptide-(hydrated anion) interaction. For example, deuterated amide III may be more sensitive than the amide II mode in terms of whether the amide ND group is hydrogen bonded or not to a hydrated anion in the neighborhood. To understand the ion effect on the amide group, molecular dynamic simulations for the NMA in single-atom ionic solution were performed. The RDFs of anion around H atom of H2O and that around amide H are shown in Figure 9a,b, respectively. It is clear that anions have a close population near the H atom of H2O, but they do not exhibit an appreciable affinity to the H atom of the amide group. The RDF of the anion around carbonyl oxygen shown in Figure 9c, on the other hand, suggests a much larger distance. Thus, the frequency shift of the amide II and amide III bands is most likely due to direct interaction between NH group and water, which will certainly be influenced by the hydrogen-bond structure of water but will only be indirectly influenced by anions that interact with water molecules. This also indicates ion effect of these ions on the structural perturbation of water exist outside the first hydration shell of the ion. In addition, as we discussed above the change of the vibrational frequency of the HOH bending mode roughly follows the classical Hofmeister anion series, except for some polyatomic anions (such as NO3− and SCN−). Further, the order of the frequency shifts of the amide II and amide III bands generally corresponds to that of the HOH bending for single-atom anionic solutions but not for some polyatomic anionic solutions, for example, Na2CO3 and Na2SO4. In Na2CO3 and Na2SO4 solutions, the vibrational frequencies of

4. CONCLUSIONS In this work, the anionic effect on the hydrogen-bonding network of water and that on the peptide−water interaction were investigated using the HOH bending mode of water and the amide I, II, and III modes of a mono peptide model NMA. The blue shift of the HOH bending frequency in kosmotropic saline solution indicates an increased water−water interaction, while the red shift of the HOH bending frequency in chaotropic saline solution indicates a decreased water−water hydrogen bonding. These observations are basically consistent with the Hofmeister anionic effect, except for NO3− and SCN− ions. In addition, except for the frequency changes of the water HOH bending mode, our work also examines the ionic effect on the spectral width and peak intensity of this mode.

Figure 9. RDFs of anion around H atom of H2O (a), anion around amide H (b), and that around carbonyl oxygen (c) in NaF, NaCl, NaBr, and NaI solutions. G


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The amide II and amide III bands are found to generally blue shift in kosmotropic saline solutions and red shift in chaotropic saline solutions. The amide I band generally shows the opposite trend in frequency as those of the amide II and III modes. Their correlated changes appear to show no extra ionic interaction on the CO group of NMA; however, high-concentration NaI causes the amide I band to blue shift its frequency more significantly than those red shifts in the amide II and III modes. Thus, care should be taken in order to quantitatively compare the sensitivity of the three amide modes to the anionic effect on peptide backbone. This is why, in our work, we compared the Hofmeister anionic effect on the amide modes under nearly identical cationic strength (Figures 7 and 8 and Table 1). Because hydrated anion and cation, in principle, can interact with both the NH side and the CO side of an amide unit. As far as the anion is concerned, its relative position to the amide NH and CO groups can be visualized using molecular dynamics simulations. Furthermore, our result also indicates that the deuterated amide II mode becomes less sensitive to peptide−anion interaction because of the isotopic effect: the potential energy distribution analysis indicates the amide II mode has less ND in-plane bending contribution than NH in nondeuterated peptide. This explains the insensitivity of the nondeuterated amide II vibrational frequency to the influence of anions.44 Finally, based on the experimental results, the order of the anionic effect on water structure and peptide−water interaction is summarized in Scheme 2. The results show that frequency shift of the HOH bending, the amide II and amide III modes in the saline solutions considered in this work are in general agreement with the order of the classical Hofmeister anionic series in a majority of cases, indicating that Hofmeister anionic series can be roughly explained by frequency shifts in water bending mode as well as amide II and III modes. The amide I mode, on the other hand, is not as sensitive as the amide II and III bands.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+86)-010-62563068. Fax: (+86)-010-62563167. E-mail: [email protected]. ORCID

Jianping Wang: 0000-0001-7127-869X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the National Natural Science Foundation of China (21603238 to J.Z. and 21573243 and 21327802 to J.W.).

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REFERENCES

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Direct Anionic Effect on Water Structure and Indirect Anionic Effect on

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