Article pubs.acs.org/JPCB
Uncovering the Sensitivity of Amide-II Vibration to Peptide−Ion Interactions Juan Zhao† and Jianping Wang*,†,‡ †
Beijing National Laboratory for Molecular Sciences and Molecular Reaction Dynamics Laboratory, 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, linear infrared spectroscopy was used to examine the effect of salt on the amide-II mode in a model β-peptide (Nethylpropionamide, NEPA) in its deuterated form, to reveal the sensitivity of this mode in reporting peptide−ion interactions. In comparison to the case of NEPA in water, the amide-II spectra mainly showed a red-shifted component in four typical saline solutions (NaCl, CaCl2, MgCl2, and AlCl3) examined in this work. Our results suggest that highly populated hydrated ion complexes under high salt concentration conditions destroy the hydration layer of the model peptide and result in mostly a salting-out state of the peptide. Molecular dynamics simulations suggest that the hydrated cation mainly interacts with the peptide backbone on the amide CO side, whereas the hydrated anion interacts on the amide N−H side. As the amide-II mode is mainly a combination of the C−N stretching and N−H in-plane-bending vibrations, this mode is advantageous in being responsive to ionic interaction from both the CO and N−H sides. Such a dual sensitivity should be very useful in probing the breaking and/or formation of the interamide hydrogen bond between the CO and N−H groups, which is a very important interaction involved in the solvation and stabilization, as well as folding/unfolding of proteins. 1700 cm−1 region of vibrational frequency, is a well-known and effective conformational marker of peptides and proteins. Studies of metal cation and peptide backbone interactions using the amide-I vibrational mode have been carried out.4,5,13,15,16 On the other hand, the amide-II mode mainly contains the symmetric linear combination of the C−N stretching and N−H in-plane bending. This mode is usually located in a frequency region that is 100 cm−1 lower than that of the amide-I mode. It is also believed to be a structural marker for peptides and proteins.17−27 Because the amide-I and amide-II modes in the same peptide unit are intrinsically coupled,28,29 the factors that affect the frequency and shape of the amide-I mode would also affect these vibrational characters of the amide-II mode. This should also be the case in peptide−ion interactions. Indeed, it has been reported that the interaction of the Cu(II) cation with a chemically modified peptide fibril caused the intensities of the amide-I and -II bands to change at the same time.30 However, the sensitivity of the amide-II mode to peptide−ion interactions remains less understood. In the gas phase, the peptide−ion interaction picture could be relatively easy to define because there is no bulk solvent intervention. For example, the metal cation can bind either to the carbonyl oxygen atom to show a charge-solvated binding
1. INTRODUCTION Specific ion−protein interactions play a very important role in biophysical and biochemical phenomena such as protein folding, association, stability, signal transducing, and drug targeting.1,2 Proteins are important biocomponents. The microscopic mechanism of ion and protein backbone interactions has become a very interesting research topic in recent years.3−11 In solution phases, monovalent and divalent metal cations are generally known to interact with the peptide group (CONH) and destruct intramolecular hydrogen-bonding interaction.4,5,8,12,13 The so-called Hofmeister ionic effects14 were proposed to explain the salting-in and salting-out behaviors of proteins in salted aqueous solutions, which are macroscopic descriptions of the interactions between ions and proteins. However, the detailed structural aspects of such interactions are still not fully understood. Infrared (IR) spectroscopy is a very powerful traditional method for probing the conformations of peptides and proteins in solution phases. Experimentally observed IR spectra are a collection of mostly fundamental normal-mode vibrational transitions, whose potential energy surfaces are extremely sensitive to solvation and hydrogen-bonding interaction, as well as the change of ionic strength in the vicinity of a given vibrational chromophore that is usually composed of a limited number of nuclei. In peptides and proteins, several amide modes can be used for the study of peptide−ion interactions. For example, the amide-I mode, which is located in the 1600− © 2016 American Chemical Society
Received: June 10, 2016 Revised: August 6, 2016 Published: August 18, 2016 9590
DOI: 10.1021/acs.jpcb.6b05889 J. Phys. Chem. B 2016, 120, 9590−9598
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The Journal of Physical Chemistry B case or directly to the nitrogen atom of the amide by replacing the amide proton, showing an iminol binding picture.31 In this case, the amide-II band would be an excellent vibrational probe to differentiate the two different binding patterns because in the iminol binding picture, the amide-II band will disappear. In the solution phase, because of the presence of bulk solvent, the scenario of peptide−ion interactions is completely different; thus, a mechanistic understanding of peptide−ion interactions is still under debate. Therefore, in this work, we aimed at finding out how sensitive is the amide-II mode in terms of reporting the peptide−ion interaction and whether the sensitivity is due to the interaction on the amide CO side or on the amide N−H side of the peptide amide unit. To address these questions, linear IR spectroscopy and computational methods were utilized. N-Ethylpropionamide (NEPA),32 a β-peptide model compound with a single amide unit, was used in this work. As an analogue of naturally occurring α-peptides that are composed of α-amino acids, β-peptides are composed of β-amino acids that are, however, artificial and not present in nature. The βpeptides can adopt very interesting secondary structures and thus provide very useful benchmarks for understanding protein folding problems.33 In terms of peptide−ion interactions, longchain and short-chain β-peptides have been utilized as model systems in recent years.34,35 In this work, our interest lies in understanding the molecular mechanisms of the peptide backbone and ion interactions; thus, it is advantageous to use short-chain peptides, as shown in this particular case. A single amide unit containing the β-peptide model, NEPA, is utilized, which provides sufficient space for peptide backbone and solvent/ion interaction to occur, from either the CO or N− H side or both of the amide group. In our recent work,16 the metal cation and peptide backbone interaction in deuterated NEPA (d-NEPA) was examined using the amide-I mode. Interaction between a dynamical cation/ water/anion complex and the amide group has been proposed to explain the experimentally observed band split in the amide-I mode. The coexistence of both salting-in and salting-out states of d-NEPA species was proposed. In this work, we further investigate the interaction between salt ions and the peptide backbone using the amide-II mode as a vibrational probe. This article is organized as follows. First, linear IR spectra were collected in the amide-II region for d-NEPA under various salt conditions, and then quantum-chemical computations and molecular dynamics (MD) simulations were carried out. Spectral band structures, molecular structural dynamics, and solute−solvent interactions were analyzed to obtain a microscopic picture of the dynamical interaction between salt ions and the peptide amide group.
Figure 1. Structure of d-NEPA and the first-order vibrational transitional density cubes (VTDCs) for the amide-II mode. Color coding for the VTDCs: magenta for positive and cyan for negative charge variations.
of desired salt concentrations while keeping the concentration of d-NEPA at 75 mM. One-dimensional IR (i.e., Fourier transform infrared (FTIR)) absorption spectra of d-NEPA in pure D2O solution and in various saline solutions were measured using a Nicolet 6700 FTIR spectrometer that was equipped with a liquidnitrogen-cooled mercury−cadmium−telluride detector. The samples were placed in a home-made IR liquid cell consisting of two 2 mm thick CaF2 IR-optical windows and a 50 μm Teflon spacer between them. The IR spectra were measured with 2 cm−1 spectral resolution and averaged over 64 scans. IR spectra of saline solutions at appropriate concentrations were also measured for background correction. Dry air was used to purge the FTIR spectrometer and its sample chamber during the spectral measurements. Even though the identical Teflon spacer was used each time, the thickness of the liquid sample may still vary from measurement to measurement to a certain degree. All of the IR measurements were carried out at room temperature (22 °C). 2.2. Molecular Dynamics Simulations. The dynamical trajectories of d-NEPA in different saline solutions were obtained from MD simulations.16 Briefly, the MD simulations were performed using the CHARMM force field for d-NEPA, the SPC/E model for water, and a set of force-field parameters for the ions16 studied in this work. Each MD ensemble contains one d-NEPA molecule in a cubic solvent box with an initial size of 28 × 28 × 28 Å3. The salt concentrations were 9.0, 4.6, 4.6, and 3.0 M for NaCl, CaCl2, MgCl2, and AlCl3, respectively. The nonbonded cutoff was set to 12 Å, and the particle mesh Ewald summation was used for long-range electrostatic interactions. Energy minimization was performed first for each MD simulation and then followed by a heating process from 0 K to the desired temperature in 20 ps. The equilibration run was performed for 1 ns to ensure a stable ensemble. MD simulations were finally performed using the Langevin-piston Nose−Hoover method for 20 ns with a step of 400 fs for coordinate data acquisition. The radial distribution functions (RDFs, denoted g(r)) of water oxygen and chloride ion around amide N−D were computed using the MD trajectories. The following formula was used to compute the number of particles, j, around particle i within a distance of r0
2. MATERIALS AND METHODS 2.1. Materials and IR Measurements. NEPA (C2H5CONHC2H5, 99% purity; Sigma-Aldrich) was dissolved in deuterated water (D2O) and lyophilized three times for H/D replacement of the amide NH group. This results in a deuterated form of NEPA (d-NEPA, C2H5CONDC2H5), whose molecular structure is shown in Figure 1. d-NEPA was then dissolved in D2O at a concentration of 75 mM for IR absorption spectral measurement. Concentrated sodium chloride, magnesium chloride, calcium chloride, and aluminum chloride (99.9% purity; J&K Chemical) salt solutions in D2O were added separately to the d-NEPA/D2O solution at a series
ni − j(r ) = 4πρj0
∫0
r0
r 2gi − j(r ) dr
(1)
ρ0j
where denotes the number density of particle j and gi−j(r) denotes the RDF of the ith and jth particle pair. 2.3. Quantum Chemistry Calculations. The density functional theory (DFT) at the level of B3LYP function with the 6-311++G** basis set was used to optimize the molecular 9591
DOI: 10.1021/acs.jpcb.6b05889 J. Phys. Chem. B 2016, 120, 9590−9598
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The Journal of Physical Chemistry B structure of d-NEPA. Harmonic normal-mode frequency computations, with and without the polarizable continuum model (PCM) for solvent (D2O),36,37 were carried out on the energy-minimized structures. The vibrational properties of the d-NEPA·D 2 O dimer, d-NEPA·Cl(D 2 O) 6 , and d-NEPA· MCln(D2O)m clusters, where M represents metal cation, and n and m (with n + m = 6) represent the numbers of chloride ions and water molecules, respectively, were also analyzed at the same level of theory. To examine the delocalization of the amide-II mode, the potential energy distribution (PED) of the mode was computed as described previously.38 For comparison, the PED values for d-NEPA using the Hartree−Fock (HF) and Møller−Plesset perturbation (MP2) theories with the 6-311+ +G** basis set were also evaluated. All of the calculations were performed using Gaussian 09.39 In addition, the first-order VTDCs for the amide-II mode of d-NEPA were computed by following the procedure described in recent works.40,41 The VTDCs can be used to visualize the redistribution of electron density associated with a specific molecular vibration, which can thus be used as an effective measure for the delocalization of the vibration mode.
frequency region. The amide-II mode is mainly composed of the C−N stretching and N−D in-plane bending of the amide group (COND). Computation shows that the contributions of the C−N stretching and N−D bending are ca. 0.30 and 0.10, respectively (see Table 1 for details). In D2O, the amide-II mode shows a single strong peak at ca. 1492 cm−1. A relatively weak doublet at ca. 1458 cm−1 is due to methyl rocking vibrations, whereas the peaks at ca. 1384 and 1355 cm−1 are mainly due to the out-plane-bending vibrations of the methylene groups, as suggested by the IR spectrum of nondeuterated NEPA in thin-layer H2O solution and the computational results (not shown). In saturated NaCl solution, the amide-II mode also mainly exhibits a single peak at 1492 cm−1 but with a decreased intensity. In 6 M CaCl2 solution, the amide-II band is significantly red-shifted (peaked at 1445 cm−1) and broadened. In 6 M MgCl2 solution, a clear splitting of the amide-II band is observed: in addition to a strong and red-shifted band (ca. 1454 cm−1), a relatively weak and slightly blue-shifted peak is observed. In AlCl3 solution, the amide-II band appears to be more interesting. It shows three peaks, that is, in addition to the features similar to those observed in MgCl2 solution, a blueshifted peak is observed at ca. 1519 cm−1. In addition, there are also intensity changes in the methylene and methyl vibrational bands, in the presence of salt ions, which will be described in the following section. 3.1.2. FTIR Difference Spectra. To further examine the effect of ions on the amide-II mode, difference IR spectra in each saline solution versus that in D2O were recorded and the results are also shown in Figure 2 (right column). The peak positions can be clearly seen in the IR difference spectra. In these difference spectra, a strong negative-going peak is shown at ca. 1492 cm−1, which is due to the amide-II absorption of d-NEPA in pure D2O. A major positive-going and relatively broad peak is shown at ca. 1440 cm−1, which is assigned to be the redshifted amide-II peak in the presence of salt ions. In addition, because the spectra were normalized in the frequency region of 1570−1300 cm−1, the difference spectra show a larger spectral change (in terms of intensity) in the presence of divalent and trivalent cations. In particular, a significant blue-shifted amide-II component is shown in the difference spectrum (bottom-right panel of Figure 2). Further, it is clear that the magnitude of the spectral change in the 1510−1390 cm−1 region is cationdependent, being significant in the case of divalent and trivalent cations but insignificant in the case of monovalent cations. In addition, there are two weak negative-going peaks at ca. 1384 and 1355 cm−1 also, which are due to the methylene vibrational modes. This suggests that these vibrational modes become weaker in the presence of salt ions. At the same time, the methyl vibration (with frequency at ca. 1458 cm−1) also shows a small negative-going peak (marked by a down arrow in the bottom-right panel of Figure 2), which is however not as obvious as those at 1384 and 1355 cm−1, because of the presence of a strong positive amide-II peak in the same region. One may argue that these negative-going CH3 and CH2 peaks may be artificially amplified in the difference spectra because these difference spectra were obtained by subtracting those normalized spectra shown on the left side, which, however, was done without assessing salt-concentration-dependent molar absorption coefficients of the CH3 and CH2 bands in this frequency region. However, from the data shown in the left column of Figure 2, for example, the case of concentrated CaCl2, it is clear that concentrated salt indeed weakens the CH3
3. RESULTS AND DISCUSSION 3.1. Experimental IR Results. 3.1.1. FTIR spectra. Figure 2 (left column) shows the FTIR spectra of d-NEPA in D2O in a frequency range of 1570−1300 cm−1, in the presence of various salt ions. The spectra were simply area-normalized in this
Figure 2. (Left) FTIR spectra of d-NEPA in the frequency range of 1570−1300 cm−1 in saturated NaCl, 6 M CaCl2, 6 M MgCl2, and 4 M AlCl3 (red curve), respectively. The IR spectrum of d-NEPA in pure D2O is also given in each case (black curve). Spectra are areanormalized in the frequency range from 1570 to 1300 cm−1. (Right) FTIR difference spectra in the same frequency region (the spectra of d-NEPA in concentrated salts minus that of d-NEPA in pure D2O). Important peak positions are marked by dashed lines, arrows, and/or wavenumbers in these IR spectra. 9592
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Table 1. Vibrational Frequencies, Intensities, and PEDs of the Amide-II mode of NEPA and d-NEPA Computed at Different Theoretical Levels and in Different Environments
a
species
environment
ν/cm−1
I/km mol−1
NEPA d-NEPA
gas phase gas phase PCM (water)
1539.2 1442.4 1450.2
221.2 207.7 376.9
NEPA d-NEPA
gas phase gas phase
1681.0 1577.1
403.5 265.9
NEPA d-NEPA
gas phase gas phase
1545.6 1465.6
279.3 237.8
PED/%a B3LYP/6-311++G** CNs (21), NHib (51) CNs (30), NDib (10), CCs (5), NCs (5), HCCib (8), HCCib (6), HCHob(14), OCNib(5) CNs (37), NDib (10), CCs (7), NCs (6), HCCib (10), HCHob(10), OCNib(7) HF/6-311++G** CNs (22), NHib (54) CNs (28), NDib (9), CCs (8), NCs (6), HCCib (12), HCHob(5), OCNib(7), HCCNτ (10) MP2/6-311++G** CNs (24), NHib (43), NCs (6) CNs (31), NDib (11), CCs (7), NCs (7), HCCib (9), HCHob(6), OCNib(6), HCCNτ (8)
s, stretching; ib, in-plane bending; ob, out-plane bending; and τ, torsional vibration. Contributions less than 5% were not listed.
MgCl2, and AlCl3 solutions. The concentration-dependent spectral changes in the IR spectra in each case are clearly shown. Specifically, the intensities of the amide-II mode at ca. 1492 cm−1 and the two peaks at ca. 1384 and 1355 cm−1 decrease as the salt concentration increases, whereas the intensity of the broad band at ca. 1440 cm−1 increases as the salt concentration increases. In AlCl3, the intensity of the blueshifted amide-II component increases gradually with the salt concentration. 3.2. Mode Delocalization. The above results established that the amide-II mode of d-NEPA is responsive to the concentration and type of metal cations. To further understand the nature of such interaction, two computational methods were utilized first to examine the delocalization degree of the amide-II mode. 3.2.1. PED Analysis. Table 1 shows the vibrational properties of the amide-II mode of NEPA and d-NEPA in different media, at the levels of B3LYP/6-311++G**, HF/6-311++G**, and MP2/6-311++G**. For nondeuterated NEPA, the results show that a large portion of the potential energy comes from the N− H in-plane bending motion (more than 50% at the DFT and HF levels and 43% at the MP2 level). There are also substantial contributions (ca. 20%) from the C−N stretching motion as predicted by different theories. Upon NH/ND exchange, the contribution of the N−D inplane bending becomes ca. 10%, which is significantly lower than that in the case of nondeuterated NEPA (ca. 50%). This is because the N−D in-plane bending vibrational frequency is redshifted with respect to the N−H bending and thus only slightly mixes with the C−N stretching mode. However, the PED value of the C−N stretching increases only to some extent: it is in the range of 28−37% at different theoretical levels, with and without the presence of the PCM solvent model. Other contributions are all at the level of 10% or less. Thus, the computation suggests that the major vibrations are from C−N stretching and N−D in-plane bending in d-NEPA, and the three different theories yield consistent results. It is known42 that deuteration of a N−H group results in a lowered N−D inplane-bending vibrational frequency, which also decouples this motion from the C−N stretching motion in the amide-II mode. Under such circumstances, the amide-II mode is mostly the C− N stretching. This is in agreement with the PED results shown in Table 1. In addition, the computation also shows that the amide-II is red-shifted by ca. 100 cm−1 upon the NH/ND exchange, which agrees roughly with the experimental observations in peptides (1567 cm−1 in nondeuterated NEPA dissolved in H2O (data not shown) and 1492 cm−1 in d-NEPA
and CH2 peaks. Thus, the presence of the negative-going CH3 and CH2 peaks in the difference spectra is justified to some extent. In addition, as larger spectral changes are observed in trivalent cations and a minor spectral change is observed in the monovalent cation case, whose ionic strengths are very similar, one may intuitively conclude that the spectral change is mainly related to the cationic charge effect. However, this is not entirely true; the anion also plays certain roles in the observed amide-II patterns. Evidence of this will be given in the following sections. 3.1.3. Concentration Dependence of the FTIR Spectra. Figure 3 shows the concentration-dependent FTIR spectra of dNEPA in the frequency range of 1570−1300 cm−1 in CaCl2,
Figure 3. Concentration-dependent IR spectra of d-NEPA in CaCl2 (a), MgCl2 (b), and AlCl3 (c) in the frequency range of 1570−1300 cm−1. The spectrum in D2O is shown in each panel (black curve). Spectra are area-normalized in the frequency range from 1570 to 1300 cm−1. 9593
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The Journal of Physical Chemistry B in D2O, meaning a frequency shift of 75 cm−1 upon the NH/ ND exchange). 3.2.2. VTDC Analysis. The VTDCs describe how the electron density of a molecule redistributes among atoms upon the motion of a given vibration mode; thus, it can serve as an effective measure of mode delocalization.40,43,44 The firstorder VTDC of the amide-II mode of d-NEPA in the gas phase at the level of B3LYP/6-311++G** was computed and is shown in Figure 1. Clearly, associated with the amide-II vibration, a large amount of electron density change occurs in the COND unit, which is more prominent along the N−D and C−N bonds. Minor electron density redistribution also occurs in another part of the molecule, such as in the methyl and methylene groups on the N-terminus side. Such a picture is in good agreement with PED analysis shown in Table 1. 3.3. Structural Dynamics. Classical MD simulations were carried out to better understand the structural aspect of the microenvironment of the peptide unit in d-NEPA. Our recent work16 has focused on revealing the structural details of solvent and salt species in the vicinity of the amide CO group through the amide-I mode; a dynamical cation/anion/water complex was proposed. In this section, we examine the local microenvironment of the amide N−D group of d-NEPA. The g(r) functions of the chloride ion around the amide D in each saline solution are shown in Figure 4a, along with the number
4.8, 5.0, 4.8, and 4.9 Å in the case of NaCl, CaCl2, MgCl2, and AlCl3, respectively (Figure 4c,d). This suggests the presence of a solvated chloride ion on the amide N−D side, not far from the amide N−D group. However, statistically, one of the ligands of such a hydrated chloride ion, that is, the D2O molecule, even though falling into the domain of the hydration layer of the amide N−D group, does not form or forms a very weak hydrogen bond with the amide N−D group (data not shown). This result suggests a depletion of water molecules under high salt concentration conditions from the N−D···D2O hydrogen-bonded moiety. In addition, our recent MD analysis16 suggested that there is no close contact between the amide O and chloride ion on the amide CO side. 3.4. Amide-II Frequency Shift and Intensity Change in the Presence of Ions. PED analysis indicates that the amideII mode in d-NEPA contains a significant contribution from the C−N stretching (30%, at the DFT level) and some contribution from the N−D in-plane bending (10%, at the DFT level). This suggests that at least two aspects should be considered in explaining the observed frequency change of the amide-II mode. 3.4.1. Cationic Effect. First, it is well known that in an amide unit (CONH) the double-bond character of the CO bond tends to delocalize toward the C−N bond. Our recent work also showed that the bond lengths of CO and C−N in the same amide unit are anticorrelated.18 Thus, a factor that strengthens the CO bond will weaken the C−N bond in the same amide unit. A polarized water/cation/anion complex is believed to interact with amide CO according to our recent work,16 and such a picture is also shown in Figure 5 for divalent and trivalent cations in a simplified way. Our previous MD simulations also suggested that the hydrogen bond between a water molecule and an amide oxygen can be switched on and off in a dynamical way in concentrated salt solutions because of the presence of a nearby cation complex that takes a water molecule that was hydrogen-bonded with amide CO to be one of the ligands. Such an on- and off-behavior of the water− amide-hydrogen bond was believed to be the main reason for the splitting of the amide-I band.16 In this work, quantum chemical computations were carried out to examine the effect of the above-mentioned possible water/cation/anion complex on the frequency of the amide-II mode. The results are given in Table 2. The frequency of the amide-II mode for the amide unit that is bonded by one and three D2O molecules (Figure 5, cases a−c) is also given for comparison. Note that among complexes a, b, and c, only complex c corresponds to a simplified solvation picture of NEPA with its amide unit surrounded by three hydrogenbonded D2O molecules. Complexes a and b are used to examine the effect of a single hydrogen-bonded water molecule on the amide-II mode. In addition, the frequency of the amideII mode for free d-NEPA is found to be 1442.4 cm−1 and that of d-NEPA with a distant water molecule placed ca. 4 Å away from amide CO or N−D is found to be very close to that in the case of free peptide. These results are also listed in Table 2 and are used as a reference in the following discussion. When the CO group of d-NEPA forms a hydrogen bond with one D2O molecule, the frequency of the amide-II mode increases to 1457.8 cm−1. This blue shift of ca. 15 cm−1 is caused by the effect of a single hydrogen-bonding on the amide CO group, which in turn affects the frequency of the amideII mode by shortening the C−N bond. When there are three water molecules (case c), the blue shift is the largest. The
Figure 4. Radial distributions and integrated number of particles. (a) RDFs of chloride ions around amide D in each saline solution. (b) The number of chloride ions around amide D in each case. (c) RDFs of the oxygen atom of D2O and amide D atom. (d) Number of D2O molecules around amide D in each case. The dashed line indicates that the number of chloride ions around amide D equals 1 (b) and that of water molecules around amide D equals 1 and 6 (d).
of the chloride ions Figure 4b. In each saline solution, the first peak appears at ca. 2.9 Å and the number of chloride ions in the first peak is estimated to be 0.14, 0.60, 0.36, and 0.25 in NaCl, CaCl2, MgCl2, and AlCl3, respectively. When the number of chloride ions is accumulated to 1, the distance between the amide D and the chloride ion is 5.6, 4.5, 5.0, and 5.2 Å in NaCl, CaCl2, MgCl2, and AlCl3, respectively. Such a distance is large enough to place a water molecule between the amide N−D group and the chloride ion. Indeed, the g(r) functions of the oxygen atom of water molecules around amide D showed that the first peak appears at ca. 2.2 Å in each concentrated saline solution; however, statistically one water oxygen atom appears at ca. 3.3, 3.7, 3.4, and 3.5 Å, and about six water molecules at 9594
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Figure 5. Structures of d-NEPA and its solvent neighboring species. (a and b) Hydrogen bonding with one water molecule, (c) hydrogen bonding with three water molecules, (d−f) hydrogen bonding with the MCln(D2O)m complex (n + m = 6, where n = 2 for divalent cations and n = 3 for trivalent cations) on the amide CO side (for Ca2+ (d), Mg2+ (e), and Al3+ (f)); (g) hydrogen bonding with the Cl(D2O)6 complex on the amide N−D side. The dashed lines indicate intermolecular hydrogen bonds.
(for example, Figure 5, case c). This is what was observed experimentally in Figure 2, along with a broadened amide-II band structure that is in accordance with the picture with a heterogeneous solvent environment. Such a red-shifted amideII band in all cases suggests that d-NEPA is most likely in the salting-out state. This salting-out picture corresponds to what is shown in Figure 5d−f, more or less, but with a broken hydrogen bond between CO and the nearby water molecule in each case. 3.4.2. Anionic Effect. MD simulations indicate that there is a hydrated chloride ion neither too far nor too close to the amide N−D group. Because the chloride ion is the common anion used in this work, the question we like to address first is whether a hydrated chloride ion complex in the neighborhood of this N−D group would affect the amide-II frequency. To answer this question, we examine the amide-II frequency under the influence of a hydrated chloride complex (Figure 5g) in comparison to that with a single hydrogen-bonded D2O molecule on the same side of the peptide unit (Figure 5b). The results are also listed in Table 2. It can be seen clearly that the amide-II frequency of the d-NEPA(D)·D2O dimer (1452.0 cm−1) is ca. 10 cm−1 higher than that in the case of free dNEPA, whereas that of the d-NEPA(D)·Cl(D2O)6 complex (1465.9 cm−1) is even higher (ca. 14 cm−1). Again, because a blue-shifted amide-II mode is observed clearly only in the presence of AlCl3, while a similar ionic strength is retained in each case, one can conclude that at a higher salt concentration more water molecules will also be taken away from the amide N−D group and cause a broken hydrogen bond between amide N−D and a nearby water molecule. On the other hand, the amide-II frequencies for the d-NEPA(O)···D2O and d-NEPA(D)···D2O complexes without hydrogen bond between the amide and D2O clearly show a generally unaffected amide-II frequency (Table 2). These results can serve as a reference for the dehydrated amide group in NEPA. The amide-II vibrational frequency shifts due to the binding of D2O and/or ionic complex on the CO and N−D sides of d-NEPA are further summarized in Figure 6 based on the data listed in Table 2. To summarize this section, the absence of amide-hydrogen-bonded water molecules in the vicinity of the peptide backbone, either on the amide CO side or on the N−D side, is most likely to
Table 2. Vibrational Transition Frequencies and Intensities of the Amide-II Mode of d-NEPA in Various Solvent Clusters and Hydration Ionic Clusters Shown in Figure 5a species
ν/cm−1
I/km mol−1
free d-NEPA d-NEPA(O)···D2Ob d-NEPA(D)···D2Ob (a) d-NEPA(O)·D2Oc (b) d-NEPA(D)·D2Oc (c) d-NEPA·3D2Oc (d) d-NEPA(O)·AlCl3(D2O)3c (e) d-NEPA(O)·MgCl2(D2O)4c (f) d-NEPA(O)·CaCl2(D2O)4c (g) d-NEPA(D)·Cl(D2O)6c
1442.4 1444.9 1444.1 1457.8 1452.0 1479.8 1469.6d 1468.4d 1467.6d 1465.9
207.7 207.6 202.7 195.8 198.0 108.6 156.0 117.5 121.2 157.9
a Calculated at the level of B3LYP/6-311++G**. bNon-hydrogenbonded species. cHydrogen-bonded species with corresponding structures shown in Figure 5. dThe mean of these three is 1468.5 cm−1, which is used in plotting Figure 6.
amide-II frequency becomes 1469.6, 1468.4, and 1467.6 cm−1 when there are hydrated complexes of AlCl3, MgCl2, and CaCl2 moieties, respectively, on the amide CO side. Thus, even though the effect on the C−N bond is a secondary or indirect one, the frequency blue shift is larger than that caused by the binding of a single D2O on the same side of the amide group. Note that these results were obtained from a rigid peptide− water dimer structure or a rigid peptide−cation/anion/water complex of each case, which set an upper limit of the blue shift (ca. 25 cm−1), in particular for the latter cases. Experimentally, indeed, a small blue-shifted amide-II band is shown clearly in the case of concentrated AlCl3 (Figure 2, bottom-left panel) but not so clearly in the case of divalent metal ions. This can be understood because in reality the rigid structures shown in Figure 5 hardly exist in solution. The heterogeneous nature of peptide−solvent interactions in a concentrated-salt environment will weaken the hydrogen-bonding interaction between the amide CO group and solvent water. As a consequence, and most probably at a higher salt concentration, more water molecules will be taken away from the amide CO group and leave a structural vacancy, which results in a red shift in the amide-II frequency with respect to the case of d-NEPA in D2O 9595
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predicted by the computational results in Table 2. Such solvent hydrogen-bonding effect on the amide-II mode from the peptide N−H side agrees with a previous theoretical report.45 In addition, in concentrated salt solutions, because of the large population of cations and anions, statistically, it is quite possible to have an unbalanced charge distribution in the vicinity of dNEPA, for example, with a MCln(D2O)m complex in the neighborhood of the amide CO group and a Cl(D2O)6 complex in the neighborhood of the amide N−D side. Clustered cation and anion hydration structures are known to exist in aqueous salt solution.46 The picture of the peptide−ion interaction derived from this work is in general agreement with the recent studies using the amide-I mode only.16 In this work, more significantly, a blue shift is shown in the amide-I mode, indicating a salting-out state of the peptide. This agrees with the results shown in Figure 2, in which mostly a red shift is observed in the amide-II mode. This makes perfect sense because the amide-I and II modes in the same amide unit are anticorrelated in frequency, as we discussed above. Furthermore, a red-shifted amide-I component was also observed in our recent study,16 in which the frequency change is less significant than that of the blue shift, and the most prominent red shift was found in the case of AlCl3. This observation also agrees with the experimental results shown in Figure 2 (bottom panels), that is, only in the case of AlCl3, a clear blue shift is shown in the amide-II band. Furthermore, the suggested cation−peptide interaction picture is also in general agreement with a recent work based on ab initio molecular dynamics and IR spectra of the amide-I mode of Nmethylacetamide (NMA).13 In addition, a very interesting comparison shows that, in terms of spectral sensitivity, in the presence of divalent cations, whereas the amide-I mode shows a moderate red shift, the amide-II mode shows an insignificant blue shift. This suggests that the amide-II mode is not as sensitive as the amide-I mode in reporting the salting-in state of the peptide backbone. This is because “salting-in” means a well-solvated state in which water and peptide form strong hydrogen bonds, which naturally affects more strongly the CO stretching motion (the main contributor of the amide-I mode) but not the C−N stretching motion (one of the main contributors of the amide-II mode). On the other hand, a well-solvated amide group can also form a strong hydrogen bond on the amide N−D side, whose in-plane bending vibration, however, does not contribute to the amide-I mode. This is the reason that the effect of anion on the amide-I mode is not sensitive. This is in agreement with a recent work in which insensitive anion effect on the amide-I mode was reported.47 Furthermore, it should be pointed out that the vibrational characteristics of the amide-II modes are similar in the naturally occurring α-peptides and in unnatural β-peptides. For example, our calculation shows that for the well-known α-peptide model compound NMA the major PED components of the amide-II mode are C−N stretching (17%) and N−H in-plane bending (40%) in nondeuterated NMA and 28 and 13%, respectively, in d-NMA, which are quite similar to the case of NEPA discussed above. Because of this, the results obtained in this work are expected to be generally applicable to α-peptides and proteins, whether they are in aqueous solutions or in fibril-like aggregates. However, clearly, deuteration of the N−H group would decrease the sensitivity of the amide-II mode to the N− H side of the amide unit in both α- and β-peptides. On the other hand, deuteration of the N−H group has insignificant
Figure 6. Blue shift of the amide-II vibrational frequency upon binding of a single water molecule to the amide CO or N−D side and upon binding of ionic complexes to the amide CO or N−D side. Averaged values were used in the case of cation complexes.
be the reason of the experimentally observed red shift in the amide-II frequency (Figure 2). 3.4.3. Transition Dipole of the Amide-II Mode. The computational results listed in Table 2 also show that the transition intensity of the amide-II mode varies significantly in different cases (roughly by a factor of 2), suggesting a solventdependent amide-II molar absorption coefficient. However, it should be noted that the harmonic approximation is not accurate enough to be reliably related to the experimentally observed vibrational transition intensity. Nevertheless, variation of intensity from cluster to cluster can serve as a justification of area normalization (which is a crude treatment) because we simply do not know how the absorption coefficient of this mode changes in different solvents. Also, considering the possible variation in the path length from sample to sample during FTIR spectral measurement, an accurate calibration of the spectral intensity (i.e., integrated peak area) in the amide-II region cannot be done in a straightforward way. Thus, the areanormalized spectra and difference spectra shown in Figure 2 are mainly useful in identifying peak positions. 3.4.4. Sensitivity of the Amide-II Mode to Peptide−Ion Interaction. Because the amide-II mode contains contributions from C−N stretching and N−D in-plane-bending by ca. 30 and 10%, respectively, we can reasonably conclude that the amide-II mode is sensitive to solvent and ionic interaction on the amide CO side and on the amide N−D side as well. The red-shifted frequencies of the amide-II bands experimentally observed in CaCl2, MgCl2, and AlCl3 solutions suggest that d-NEPA is mainly in the salting-out state. On the other hand, the appearance of a blue-shifted component, clearly shown in the case of AlCl3 saline solution, suggests that d-NEPA is also in the salting-in state partially. This can be explained using a dynamical cation complex in the vicinity of the amide CO group, which is yet to be verified by experiment. Because the frequency shift of amide-II is anticorrelated with that of the amide-I mode, the ionic effect, although partially being an indirect one (affecting C−N via CO), appears to be effective too because the amide CO group serves as a very sensitive “frontier group”, which is a very important polar group in interacting with polar solvent molecules and charged species. Such a secondary impact is the partial origin of the frequency sensitivity of the amide-II mode, whose transition dipole is known to be closely dependent on the interpeptide charge flux,17 which is presumably sensitive to the solute−solvent interactions. This is also why the computed transition intensity of the amide-II mode varies from one case to another in Table 2. On the other hand, water hydrogen-bonding on the peptide N−D side also affects the amide-II vibrational frequency, as 9596
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dynamic switching between the salting-in and salting-out states of the same peptide amide in concentrated salt solutions, ultrafast two-dimensional IR spectroscopic studies48−50 are undergoing in our laboratory. Furthermore, the MD simulations can be used to sample instantaneous peptide structures under various salt conditions, on which the amide-II vibrational frequencies of peptides can be theoretically evaluated. This may help one gain more insights into the peptide−ion interactions and better understand the probing power of the amide vibrational modes.
influence on the vibrational nature of the amide-I mode (mainly CO stretching with PED of ca. 81−84%) for both NMA and NEPA in their nondeuterated and deuterated forms (data not shown). This suggests that a combination of the amide-I and -II bands should work more effectively in both α- and β-peptides, in terms of probing ion−peptide backbone interaction, which is the main focus of this work.
5. CONCLUDING REMARKS In this work, the interaction of peptide and salt ions was examined using IR spectroscopy and computational chemistry methods. Specifically, the amide-II mode has been used as a spectroscopic probe. The vibrational frequency and line shape of the amide-II band of a model β-peptide (NEPA) in its deuterated form are found to be very sensitive to the chemical environment of the vibrational chromophore, that is, the amide unit. FTIR spectra showed mainly a red-shifted component in the amide-II mode in the presence of four saline solutions examined in this work (NaCl, CaCl2, MgCl2, and AlCl3) and a blue-shifted component more clearly in the presence of the trivalent metal cation. The vibrational characteristics of the amide-II mode were dissected in terms of its PED and the first-order VTDCs. Computational analysis on the hydrated cation and anion clusters and their influences on the amide group suggest that the red-shifted amide-II component experimentally observed is mainly due to the lack of water molecules in the vicinity of the amide group at high salt concentrations. Such an effect on the amide-II mode is significant for a hydrated cation complex interacting with the amide CO group and also significant for a hydrated anion complex interacting with the amide N−D group. However, the former effect is an indirect influence on the C−N stretching, whereas the latter is a direct influence on the N−D in-plane bending, which are the two major components of the amide-II vibration in peptides. Because the PED weights differently for the C−N stretching and N−H in-plane bending (ca. 20 and 50% for nondeuterated NEPA and ca. 30 and 10% for deuterated NEPA, respectively), both vibrations are sensitive to peptide−ion interactions. They form the basis of the structural sensitivity of the amide-II mode to peptide−ion interactions. These results, together with our recent study using the amide-I mode, provide a more detailed microscopic picture on the ionic influences on the peptide backbone, which is applicable to naturally occurring α-peptides as well. Also, it should be pointed out that so far the results presented in this work were obtained on a peptide model compound with only one peptide unit. It would be interesting to investigate ion−backbone interactions as ion−side chain interactions for both neutral and charged peptide oligomers as well, which is currently undergoing in our laboratory. In summary, the amide-II mode is effective in reporting peptide backbone and ion interactions. Even though it appears to be somewhat less sensitive than the amide-I mode, it shows the advantage of being sensitive to the ionic influences on both the amide CO and N−H sides of the backbone amide group. On the contrary, the amide-I mode is mostly sensitive to the peptide−ion interaction on the amide CO side. The dual sensitivity of the amide-II mode should be very useful in probing the breaking and/or formation of the interamide hydrogen bond between the CO and N−H groups in peptides and proteins, which is a very important interaction (both intra- and inter-molecularly) involved in the solvation and folding/unfolding of proteins. In addition, to verify the
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: (+86)-010-62656806. Fax: (+86)-010-62563167. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was supported by the Hundred Talent Fund of the Chinese Academy of Sciences and by the National Natural Science Foundation of China (21173231, 30870591, and 21573243). J. Shi contributed to early FTIR spectral measurement presented in this work. The financial support to J.W. from the State Key Laboratory of Precision Spectroscopy of East China Normal University (Open Research Fund) was also acknowledged.
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