Selectively Probing the Structures and Dynamics of Î²-Peptide

Article pubs.acs.org/JPCB

Selectively Probing the Structures and Dynamics of β‑Peptide Aggregates Using the Amide‑A Vibrational Marker Jianping Wang,* Fan Yang, and Juan Zhao Beijing National Laboratory for Molecular Sciences, Molecular Reaction Dynamics Laboratory, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ABSTRACT: The N−H stretching vibration in a β-peptide model compound, N-ethylpropionamide (NEPA), was characterized by one-dimensional infrared (1D IR) and two-dimensional (2D) IR experiments and ab initio anharmonic frequency computations. A narrowband pump−broadband probe 2D IR method was applied to selectively probe a subensemble of the N−H stretching vibrations from a mixture of different NEPA molecular aggregates that were formed via an intermolecular hydrogen bond. Vibrational lifetime and anharmonicity were found to be sensitive to the aggregation ensembles. In particular, diagonal anharmonicities were observed experimentally and confirmed computationally to be smaller for NEPA trimer than for dimer, which was explained by the presence of non-negligible off-diagonal anharmonicities in coupled N−H stretching modes.

I. INTRODUCTION The NH group works as a well-known spectroscopic probe for peptides and proteins1 because it is part of the amide unit (−CONH−) that periodically appears in the backbone of polypeptide. The N−H stretching vibration is known to be exclusively a localized mode, even when the NH group is hydrogen-bonded with, for example, the CO group of a different amide unit, thus enabling a very effective local probe for peptide structures and dynamics. Extensive experimental and theoretical studies have been reported on the N−H stretching mode in peptides and other biomolecules, as well as in small organic molecules, using steady-state and time-resolved infrared (IR) spectroscopic methods.2−18 N-Ethylpropionamide (NEPA) is an interesting model molecule of β-peptide19−22 that contains a single peptide unit.23−25 Understanding the vibrational properties of single peptide unit through the amide N−H stretching mode, in particular, allows one to get more insight into the structural and dynamical behavior of β-peptide backbone. There are advantages to studying NEPA as a model β-peptide, one of which is that it allows a direct solvent access to the amide unit, thus proving an interesting case study on amide−solvent interactions.25 We have recently studied the amide-I mode of NEPA in aqueous media using both linear IR and nonlinear two-dimensional (2D) IR spectroscopies, and the vibrational spectral diffusion dynamics of the amide-I mode have been obtained,24 which were found to resemble those observed in an isolated model amide unit for the α-peptide, i.e., N-methylacetamide (NMA).26 Aggregation of peptide oligomers is very useful in molecular assembling and novel macromolecular structure fabrication. Peptide oligomers often serve as a starting point for fabricating © XXXX American Chemical Society

well-organized macro-assemblies or forming misfolded structures. Thus, it is of great importance to examine the structural and dynamical aspects of peptide oligomers. In a very recent work, we have examined the aggregation behavior and the structural dynamics of NEPA in various solvents using the N−H stretching mode (also called the amide-A mode). Broadband pump−broadband probe IR spectroscopy was used. Solute−solute hydrogen-bonded and solute−solvent bonded complexes have been observed in nonpolar solvents.25 In particular, we found that in a weak polar solvent (CHCl3) there were two overlapping amide-A components that indicate two different NEPA aggregation states. However, due to limited structural resolving power of the broadband pump−broadband probe method, the exact aggregates were not identified. In this work, a narrowband pump pulse is utilized, aiming to further resolve the aggregated species. Collecting 2D IR spectra using a narrowband pump− broadband probe scheme has been known to be very effective and convenient,27−32 which is a frequency-domain method. In the past decade, the method of acquiring 2D IR spectra has been extended to vibrational echo-based and time-domain method, with working frequency covering a wide mid-IR range, and has been used to obtain molecular structures and dynamics information on ultrafast time scales.33−43 However, from a spectroscopic aspect, the narrowband pump−broadband pump IR method has at least one advantage: because it is a frequencyReceived: October 20, 2015 Revised: November 22, 2015

A

DOI: 10.1021/acs.jpcb.5b10249 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

principle, a typical single frequency response at a specific narrowband excitation can be deconvoluted to better determine the time zero, which was found to be ca. 300−500 fs earlier than the time point of maximum pump−probe signal. However, since the 2D IR spectral feature of NEPA does not vary much in the time window of first picosecond starting from the signal maximum, no time correction was applied. All nonlinear IR measurements were carried at room temperature (22 °C). Molecular Dynamics Simulations. Classical MD simulations were performed using the CHARMM force fields for solvent CHCl3 and the force fields from previous works for NEPA.23,24 The MD ensemble contains one NEPA and 188 CHCl3 molecules in a cubic unit cell (28 × 28 × 28 Å). The MD simulations were performed under NPT (constant number, constant pressure, and constant temperature) condition with a targeted pressure of 1 atm with periodic boundary conditions applied. A 12 Å sphere cutoff distance and a 13.5 Å pair-list distance were applied to the van der Waals and short-range electrostatic interactions, respectively.44−46 The particle-mesh Ewald summation47 was used for long-range electrostatic interactions. The Langevin piston Nosé−Hoover48 method was used for simulation in the isothermal−isobaric ensemble. A 10 ns MD simulation was performed at 298 K, and trajectories were extracted at every 200 fs. NAMD48 software was used for the MD simulations. Quantum Chemical Calculations. Structural optimization and anharmonic frequency calculations were carried out for NEPA monomer, homodimer, and homotrimer in gas phase. For the NEPA homodimer, the initial distance between the NH group of one NEPA and the CO group of another NEPA was set to 2.0 Å and finally optimized to 2.0 Å. For the NEPA homotrimer, the initial distance between NH group for the NEPA of interest and CO group of another NEPA molecule was set to 2.0 Å and finally optimized to 1.95 Å. Geometry optimization and frequency computation were carried out at the level of B3LYP/6-31+G*. Potential energy distribution (PED) functions were evaluated using VEDA.49 Anharmonicity composition analysis for the N−H stretching modes was carried out by following our previous work.50 Vibrational Transition Density Cubes. The first-order vibrational transition density cubes (VTDCs) for the NH mode of NEPA in monomeric and dimeric forms was computed by following the procedure in our recent work.51 The VTDC visualizes how the electron density of a molecule changes during the action of a given vibrational motion,52,53 which can be used to reveal the mode delocalization degree:52

domain method, it can selectively excite a subset of molecular species if it possesses a unique IR absorption band. In this work, we focused on the amide N−H stretching mode of NEPA solvated in a weak polar solvent (CHCl3), using mainly a narrowband pump−broadband probe IR method for the study of the vibrational dynamics of biomolecules in the 3 μm wavelength region. Linear IR spectroscopy and computational methods were also used. Molecular aggregation state dependent vibrational characteristics of the N−H stretching modes were investigated. The microscopic picture of solute− solvent interactions and structural dynamics of NEPA was examined by molecular dynamics (MD) simulations.

II. EXPERIMENTAL METHODS Materials and FTIR Measurement. N-Ethylpropionamide (NEPA, 99% purity) was purchased from Sigma-Aldrich and used without further purification. Solution sample was prepared in CHCl3, which is one of the common solvents used that permits the study of N−H stretching vibrations using IR spectroscopy. The concentration is 0.45 mol L−1 in order to form moderate population of dimeric NEPA. FTIR spectra in the N−H stretching region were measured using a 6700 FTIR spectrometer (Nicolet) with 2 cm−1 resolution for 64 scans. A liquid nitrogen-cooled mercury−cadmium−telluride (MCT) detector was used. A homemade IR sample cell consisting two 2 mm thick calcium fluoride IR plates that are separated by a 50 μm Teflon spacer was used. Constant dry air purging was used during spectral measurement. All FTIR spectral measurements were carried out at room temperature (22 °C). Infrared Pump−Probe Experiments. An IR pump− probe experiment was carried out using a home-built femtosecond nonlinear IR spectrometer. A Ti:sapphire regenerative laser amplifier with 800 nm central frequency, sub-35 fs pulse width, 3 mJ pulse energy, and 1 kHz repetition rate was used to pump a home-built optical parametric amplifier (OPA) based on potassium titanium phosphate (KTiOPO4, KTP) crystal. The OPA generated a near-IR signal and a midIR idler beam. The tunning range for the idler was 1.6−4.5 μm, with a typical spectral width (full width at half-maximum, FWHM) of 250 cm−1 and pulse energy of 8 μJ. This mid-IR pulse was split into a pump and a probe pulse with an 85:15 CaF2 beam splitter. The probe pulse was further attenuated not to saturate a 32-element MCT array detector. The time delay between pump and probe pulses was controlled by a translation stage with tens of femtoseconds time step. Two laser pulses were focused on sample by a 10 cm focal-length parabolic mirror. A half-wave plate and a polarizer were used in the optical path of the pump pulse to control its power and polarization. In addition, the vibrational population relaxation was measured by broadband pump and broadband probe IR method at the magic-angle polarization condition; however, kinetic traces were analyzed only at desired frequency positions. For narrowband pump−broadband probe IR experiment, an IR optical interferometer was applied on the pump pulse and narrowed its spectral width down to typically 15 cm−1 (FWHM). By varying the pump frequency and recording the probe signal, a 2D IR spectrum can be obtained at parallel polarization condition for a series of delay times. An IR monochromator with a 100 lines mm−1 grating was used to disperse transient IR signal on to the MCT array detector, resulting in a spectral resolution of 12 cm−1. For simplification 2D IR data were presented in such a way that zero delay time was set to when maximal 2D IR response was observed. In

∂D/∂Q i = [D(δQ i) − D( −δQ i)]/2δQ i

(1)

Electron density cube (D) is computed at two molecular structures for a given vibrational mode (the ith mode) with normal coordinate (Qi) varied by two a small step in opposite direction (δQi and −δQi). The electron density cube was computed for the two new structures. The obtained D(Qi + δQi) and D(Qi − δQi) were used to compute the VTDC using eq 1. Geometric optimization and frequency analysis were first performed to obtain the equilibrium structure and the normal coordinates. In this work, the vibrational step was set to 0.01 Å amu1/2 for the amide-A normal mode of NEPA. The geometric optimization, vibrational calculation, and charge analysis were carried out using Gaussian09.54 The electronic density cubes were computed using Multiwfn.55 The VTDCs were obtained on a cubic grid with totally 1003 sampling points. B


Article


III. RESULTS AND DISCUSSION Multiple Aggregates. The IR absorption spectrum of NEPA solvated in CHCl3 is given in Figure 1. It has been

Figure 2. VTDCs of the amide-A mode in NEPA of free NH group in monomer (left), of hydrogen-bonded NH in dimer (middle), and that in trimer (right).

Table 2. Computed Anharmonic Vibrational Transition Frequency (ν), Anharmonicity (Δ), Transition Intensity (I), Bond Length (rNH), and PED of the N−H Stretching Mode of Free NH in NEPA Monome, and Those of HydrogenBonded (HB) NH in NEPA Dimer and Trimer

Figure 1. Linear IR absorption spectrum of NEPA in CHCl3 in the N−H stretching region.

shown in our recent study25 that at least three different NEPA species exist in this solvent. Starting from the high-frequency side, the sharp peak at 3450.0 cm−1 is due to NEPA monomer (component a), which is a solvated monomer, or can also be called heterodimer (NEPA-CHCl3). A broad peak at 3320 cm−1 is found to contain at least two components by transient IR spectroscopy.25 This broad peak is likely to be due to hydrogen-bonded NEPA dimer (peaked at 3340 cm−1, component b) and NEPA trimer (or larger oligomer, peaked at ca. 3290 cm−1, component b′). For simplification, we only mention the trimeric contribution in component b′ in this paper. It should be noted that component a also contains the N−H stretching vibration from terminal NH groups of NEPA oligomers. Peak position, FWHM, and peak area of the IR peaks and their assignment are listed in Table 1. Concentrationdependent FTIR measurement supports the band assignment.25 In addition, a weak band appearing at 3200 cm−1 could also be due to overtone or combination bands.

peak position (cm−1)

peak width (cm−1)

area (arb. units)

a b b′

3450 3340 3290

20 65 62

14 43 42

monomer

HBdimer

HBtrimer

−1 νanhar NH (cm ) −1 ΔNH (cm ) I (km/mol) rNH (Å) PED

3444 152a 17 1.011 1.00

3352 178 437 1.018 0.99

3334 162b 568c 1.019 0.92

a

Result also applies for the terminal N−H stretching mode. bAveraged value for two hydrogen-bonded N−H stretching modes, with an offdiagonal anharmonicity of 52 cm−1. cAveraged value for two hydrogenbonded N−H stretching modes.

anticorrelated with the frequency, suggesting the stronger the hydrogen bond, the lower the N−H bond order. Moreover, transition intensity is enhanced quite dramatically in dimer and trimer as a result of hydrogen bond formation. The intensity information can be used to estimate the relative population of NH groups (i.e., the population of free NEPA (or NEPA− solvent heterodimer) and of hydrogen-bonded NEPA oligomers) from the FTIR spectrum shown in Figure 1. This can be done by taking the averaged intensity of hydrogenbonded N−H stretching mode in NEPA dimer and trimer and by assuming the free N−H stretching modes (of monomer and of the ending NEPA molecule in an oligomer). The ratio of the populations of free NEPA and of hydrogen-bonded NEPA is estimated to be 6.7:1. This means in the weak polar solvent such as CHCl3 the total amount of dimer and trimer (and larger oligomers) is only ca. 13%, and the remaining 87% is heterodimer (or “free NEMA”) and terminal NEPA molecules in oligomers with free NH. Note that the transition intensity of hydrogen-bonded N−H stretching mode in NEPA tetramer on average is quite similar to that in trimer, so excluding larger aggregates does not change the main conclusion here. Moreover, Table 2 shows that the N−H stretching mode is highly localized for both free and hydrogen-bonded NH groups. The localization degree only slightly decreases as the size of NEPA aggregates increases. Such a localized picture can also be visualized using VTDCs, and the results are given in Figure 2. Clearly, the transition charge density is localized on the NH group in both monomer and dimer but is slightly delocalized onto a neighboring hydrogen-bonded NH group in trimeric species. This delocalization manifests the intermode

Table 1. Peak Position, Peak Width (FWHM), and Peak Area of the N−H Stretching Vibrations of NEPA and Their Assignments componenta

NEPA species

a

Assignment: monomer and free NH (a); hydrogen-bonded NH in homodimer (b), and those in homotrimer and/or oligomers (b′).

Vibrational Properties of the N−H Stretching Modes. To explain the observed FTIR spectrum, NEPA monomer, homodimer, and homotrimer were considered (shown in Figure 2), and the calculated anharmonic N−H stretching vibration frequency, anharmonicity, and potential energy distribution (PED) functions of free or hydrogen-bonded NH in gas phase are listed in Table 2. The gas-phase value of the N−H stretching vibration frequency in NEPA monomer is 3444 cm−1, while those of hydrogen-bonded NH in NEPA homodimer and homotrimer are 3352 and 3334 cm−1, reasonably reproducing the experimental results shown in Table 1, mainly in the order of frequency positions but not in actual frequencies and their separations. The N−H bond length is found to be C


Article


Figure 3. Intensity-normalized narrowband pump−broadband probe 2D IR spectra of the N−H stretching vibration of NEPA in CHCl3 at selected delay times. Diagonal lines are shown in dashed lines.

components are better resolved in the projection plot than in the linear IR spectrum shown in Figure 1 because the former is proportional to the fourth power of the vibrational transition dipole while the latter is only proportional to the square of the transition dipole. The orientation of the 2D peaks and their changes as a function of the delay time between the pump and probe pulses are important spectroscopic measures for ultrafast structures and dynamics. For an isolated anharmonic oscillator, meaning no frequency overlap with any other oscillators in frequency, simply by extracting the time-dependent tilting of 2D IR signal one can obtain information about the frequency timecorrelation function (FCTF), which is a measure of spectral diffusion.26 In principle, one can obtain the autocorrelation function for the 0−1 and 1−2 transitions independently; one can also obtain the cross-correlation function for the 0−1/1−2 transitions. However, because components b and b′ are overlapped heavily in frequency, it is not straightforward to extract the time-dependent tilting information or any other prevailing methods26,56−59 for such a purpose. Thus, the spectral diffusion dynamics is not characterized in this work. Nevertheless, the time constants of the spectral dynamics for both components are estimated to be between 1/2 and 1 ps because at 1.2 ps both the blue and red signals are near vertically orientated. Such a time constant likely reflects the dynamics of weak hydrogen-bond forming and breaking between the amide unit and solvent (i.e., N−H···Cl−C bond). Aggregation-Dependent Anharmonicity. The anharmonic separation between the 1−2 and 0−1 transitions is clearly shown in the delay-time dependent 2D IR spectra (Figure 3), which provides a direct measure of the anharmonic shift of the 1−2 transition energy with respect to the 0−1 transition, i.e., the diagonal anharmonicity. Fitting a slice along the probe frequency at a pump frequency where maximum 2D signals appear, one can obtain the diagonal anharmonicity. Figure 5 shows such slices for the high-frequency component (b) with νpump = 3373 cm−1 (near the half-height of the component at the high-frequency side) and for the lowfrequency component (b′) with νpump = 3264 cm−1 (near the half-height of the component at the low-frequency side). These pump frequencies were chosen to minimize the overlap of the two components. A pair of negative and positive signals is fitted by two Gaussian functions. For component b, the anharmonicity is determined to be 131 cm−1, while for component b′,

coupling, which is in agreement with the noteworthy offdiagonal anharmonicity (52 cm−1 in this case). Dynamical 2D IR Spectra. Figure 3 shows the delay-timedependent narrowband pump−broadband probe 2D IR spectra of the N−H stretching vibration of NEPA in CHCl3. At each delay time, there is apparently a pair of strong 2D IR signals. The blue signal is attributed to the n = 0 to n = 1 transition (with n being vibrational quantum number, simply denoted as the 0−1 transition), while the red signal is attributed to the n = 1 to n = 2 transition (simply denoted as the 1−2 transition). By comparing the 2D IR profile to that of the linear IR spectrum shown in Figure 1, one can see that this frequency region contains actually two IR components (b and b′) that are likely to be due to NEPA dimer and trimer, respectively, as also confirmed in our recent work.25 Thus, in each panel the blue and red 2D IR signals shown in Figure 3 are actually composed of two pairs of signals. One pair is due to component b at the high-frequency side with a blue signal coming from the 0−1 transition that is peaked at νprobe = 3340 cm−1 and a red signal coming from its anharmonically shifted counterpart. The other pair is due to component b′ located in the low-frequency region with a blue signal peaked at νprobe = 3290 cm−1 for the 0−1 transition and a red signal that is anharmonically shifted along the probe frequency. The presence of the components b and b′ is easily seen in a projection plot (Figure 4), which is the integration of the red signal only for a range of νprobe frequency onto the νpump frequency axis. One can see that these two

Figure 4. Projection of the 1−2 transition on the pump-frequency axis. Delay time is averaged from 200 to 500 fs, and the frequency integration range of νprobe is 3125−3182 cm−1. D


Article


Figure 5. Anharmonic fitting of the N−H stretching mode in NEPA. A slice of 2D IR spectrum along the probe frequency is chosen at (a) νpump = 3373 cm−1 (near the high-frequency side of half-height of component b) and (b) νpump = 3264 cm−1 (near the low-frequency side of the half-height of component b′). Spectra were chosen at the maximum pump−probe 2D IR signal.

the anharmonicity is only 91 cm−1, suggesting that going from the dimer to trimer (or larger aggregates) the anharmonicity of the hydrogen-bonded N−H stretching becomes somewhat smaller. In other words, the diagonal anharmonicity is aggregation state dependent. In addition, by taking similar 2D IR spectral slices at later delay time (e.g., 1.0 ps, data not shown) and comparing them to those at 200 fs, a quite similar spectral profile was found for each component (b and b′). This suggests that the fluctuation of anharmonicity on the picoseconds time scale is insignificant. To understand the observed change in anharmonicity, ab initio computations were carried out, and the results are listed in Table 2. It shows that the diagonal anharmonicity first increases dramatically from NEPA monomer to dimer and then decreases from dimer to trimer. The increased diagonal anharmonicity is obviously due to intermolecular hydrogenbonding interaction, while the further decrease in the anharmonicity in trimeric NEPA is because of the appearance of appreciable off-diagonal anharmonicity. For example, in NEPA trimer there are two hydrogen-bonded N−H stretching modes, which are strongly coupled and whose off-diagonal anharmonicity is predicted to be 52 cm−1. We also further examine the origin of the diagonal anharmonicity of the N−H mode in NEPA. Figure 6 shows computational analysis of the composition of the diagonal anharmonicity of the N−H stretching modes in free NH (monomeric NEPA or terminal NH in oligomeric NEPA) and in hydrogen-bonded NH. These plots reflect the contribution from the diagonal cubic and quartic force constants of a given N−H mode, which are dominant in all cases. The plots also reflect contributions from off-diagonal cubic and quartic force constants of a given N−H mode and other normal modes, which are much less significant except for nearby N−H modes in oligomer cases. In the case where more than one hydrogenbonded NH group exist (e.g., NEPA trimer), the off-diagonal anharmonicities must be non-negligible and the diagonal anharmonicities will be smaller, which is clearly shown in Figure 6. In addition, the figure also clearly shows the intrinsically localized nature of the N−H stretching modes. To discuss this phenomenon more generally, for a given set of coupled anharmonic oscillators the total anharmonicity (diagonal and off-diagonal ones) is roughly a constant. This has been shown to be the case for coupled amide-I mode in αpeptides.60 Thus, depending on the strength of coupling, the off-diagonal anharmonicity may increase significantly at the cost of diagonal anharmonicities. If the coupling is negligibly small, then the diagonal anharmonicity will remain unaffected.

Figure 6. Diagonal anharmonicity composition analysis for the N−H stretching modes in monomeric and oligomeric NEPA. (a) Free NH group. (b) Hydrogen-bonded NH group in NEPA dimer. (c, d) Two NH groups in hydrogen-bonded NEPA trimer (HBs are defined in Figure 2). The unit of anharmonicity is in cm−1.

Further, these theoretical analysis and anharmonicity measurements suggest that component b is definitely not due to NEPA dimer, but rather due to NEPA trimer or very short oligomers. Also, the computations suggest that given the same integrated linear IR absorption area, the population of trimer (or larger oligomers) is less than that of dimer because the latter has relatively larger transition dipole magnitude (Table 2). Even larger NEPA aggregates might be much less populated in CHCl3 at the concentration condition used in this study (0.45 mol L−1). The anharmonicity can also be measured using broadband pump−broadband probe transient IR spectra. In our recent study,25 broadband pump−broadband probe IR results have been used to obtain the anharmonicity of the N−H stretching mode in NEPA solvated in CHCl3. It turned out that because of the spectral overlap of the components b and b′, and also because of experimental uncertainties in the transient IR signal, an accurate determination of the anharmonicity is not possible. It was estimated that the anharmonicity for the component b is 101 cm−1, and that for the component b′ is 110 cm−1, with ca. 12% experimental error. Here reported values, 130 and 91 cm−1, respectively, are more reasonable because the two E


Article


These lifetimes are slightly longer than that reported in a hydrogen-bonded N−H stretching mode in proline dipeptide dimer solvated in CHCl3 (0.75 ps).61 The T1 time is a measure of how fast the vibrational energy dissipates through intramolecular vibrational energy redistribution (IVR) and intermolecular vibrational energy relaxation (EVR).62 Given the solvent situation, difference in T1 time reflects structural difference in solute and/or solute−solvent complexes. In NEPA−NEPA dimer, an intermolecular hydrogen bond is formed, which may be weaker than the intramolecular hydrogen bond presented in proline dipeptide, so the IVR is not efficient in the former and eventually T1 is slower. On the other hand, for NEPA trimer/oligomer, the NH group of interest is hydrogen-bonded to the CO group of a neighboring NEPA molecule, whose amide unit may have two hydrogen bonds; its NH is also hydrogen-bonded to a neighboring CO. Thus, a more efficient energy relaxation channel for the excited NH is established in trimer or larger sized oligomer. Solvation Dynamics. Radial distribution function (RDF), also denoted as g(r), computed from the MD simulations can reveal how solvent molecules are distributed in the vicinity of the NH group. The RDFs of the NH group and nearby solvent moieties in CHCl3 are shown in Figure 8. For weak polar

spectral components are better resolved along the pump frequency (Figure 3) and also because the decreased anharmonicity for component b′ is supported by computational analysis presented above. In addition, the anharmonicity of the free N−H stretching of NEPA solvated in CHCl3 was determined to be 124 cm−1,25 which is smaller than those found in NEPA dimer and trimer. The increased anharmonicity upon intramolecular hydrogen bond formation is well-known and has also been observed experimentally previously, for example, in proline dipeptide (170 cm−1),61 in a model dipeptide AcAlaOMe (144 cm−1), and in caprolactam (148 cm−1).29 These results also suggest that strong intramolecular hydrogen-bonding interaction can affect more heavily on the diagonal anharmonicity of the N−H stretching mode. Lifetime of the N−H Stretching Vibrational States. Broadband pump and single-frequency probe was used to extract the vibrational dynamics of the N−H stretching mode of NEPA in the forms of monomer, dimer, and trimer. Magicangle polarization between the pump and probe pulses was used to avoid orientational contribution in the vibrational relaxation dynamics. The vibrational relaxation time, also called vibrational lifetime (T1), was obtained at the maximum frequency position of the 0−1 transition (bleaching of the vibrational ground state) for each IR spectral component. The results are shown in Figure 7, and the fitting results are listed in Table 3.

Figure 8. Radical distribution functions between the NH group of NEPA and various atoms in CHCl3.

solvent such as CHCl3, there is no preferential solvent−solute contact with the NH group (main peak appeared at 5.5 Å or above). Even though the molecular polarization of CHCl3 is along the C−H axis, one of the Cl atoms may form weak hydrogen bond with the NH group. Thus, a weak peak appears at ca. 3 Å (dotted curve in Figure 8). These results indicate that when NEPA dissolves in CHCl3, solvent molecules are randomly distributed near the NH group with no close contact during the most of MD times; and NEPA and CHCl3 may form weakly bonded heterodimer (i.e., solvated monomer) with quite low probability. This MD result is a low-concentration limit. As the concentration of NEPA increases, self-aggregates, including dimer, trimer, and larger sized oligomers, will form in this weak polar solvent, whose population is NEPA concentration dependent. This picture is in agreement with the IR spectrum shown in Figure 1, in which different forms of NEPA were observed. Broadband and Narrowband IR Pump−Probe Methods. Using the broadband pump−broadband probe IR method in the N−H stretching frequency region,25 our recent work showed that for NEPA solute−solute hydrogen-bonded complex and solute−solvent bonded complex coexist in nonpolar solvents. In CHCl3, two amide-A IR bands were found to heavily overlap with one another, however, still showing two different NEPA aggregation states. These results

Figure 7. Vibrational lifetimes of amide-A mode of NEPA in CHCl3 evaluated using the ground-state bleaching signals. Probing frequencies are 3452 cm−1 for free NH, 3340 cm−1 for hydrogen-bonded dimer, and 3310 cm−1 for hydrogen-bonded trimer and/or larger oligomers.

Table 3. Exponential Fitting Parameters for Magic-Angle Pump−Probe Signals of the N−H Stretching Mode of NEPA Solvated in CHCl3 component

probe freq (cm−1)

A (%)

T1 (ps)

assignment

a b b′

3452 3340 3310

65 100 100

4.7 1.4 1.0

monomera dimer trimer

a

There is an unlisted fast component with T1 = 0.7 ps and A = 35%. Contributions from the free N−H stretching in NEPA oligomers are also included in this component.

It was found that the T1 time of component a (free N−H stretching mode) is ca. 4.7 ps (the slow component), and that of component b (the hydrogen-bonded NH in NEPA dimer) is ca. 1.4 ps, while that of component b′ (the hydrogen-bonded NH in NEPA trimer) is some what shorter (ca. 1.0 ps). There is also a less significant fast component for component a, which could be due to the fact that this component actually contains contributions from free NH in the hydrogen-bonded oligomers. F



■

demonstrated the limited structural resolving power of the broadband pump−broadband probe method. In the present work, the narrowband pump−broadband probe 2D IR method was utilized so that a subensemble of the N−H stretching vibrations in the mixture of different NEPA aggregates can be spectrally selected and examined.

Article

AUTHOR INFORMATION

Corresponding Author

*Tel (+86)-010-62656806; Fax (+86)-010-62563167; e-mail [email protected] (J.W.). Notes

The authors declare no competing financial interest.

■

IV. CONCLUDING REMARKS In this work, the dynamical and structural information on the βpeptide model compound N-ethylpropionamide (NEPA) in the form of N−H stretching vibrations was obtained by using the infrared pump−probe method. Narrowband pump−broadband probe 2D IR spectroscopy of NEPA in the N−H stretching region was examined. Such a narrowband pump−broadband probe approach was used effectively in isolating a subset of molecular aggregates and studying the vibrational and structural dynamics of each subensemble. Linear IR and 2D IR spectroscopic results provided direct evidence for the presence of solvated NEPA monomer, NEPA− NEPA dimer, and trimer. Frequency analysis suggested a total amount of dimer and trimer of NEPA is only of ca. 13% in population in the weak polar solvent (CHCl3) at a moderate concentration (0.45 mol L−1). Of course, some larger oligomers might also be present in the low-frequency component b′, although with a very limited population. The N−H stretching mode is highly localized in monomeric or hydrogen-bonded homo-oligomeric structures, as shown by vibrational transition density cube analysis, potential energy distribution analysis, and diagonal anharmonicity composition analysis. The vibrational lifetime of the excited N−H stretching vibration was found to be aggregation state dependent, reflecting different vibrational relaxation pathways. MD simulations further provide a microscopic solvation picture for NEPA in CHCl3, suggesting that in most cases NH group does not interact with strongly with solvent. In a sense that solvent works as a dilution media for NEPA. This is also why solute−solute aggregates can form at higher concentration conditions. On the other hand, the diagonal anharmonicity of the N−H stretching mode was also found to differ in different aggregation species by ab initio computations. In particular, a large increase occurs from free NH species to hydrogen-bonded NH species, and a substantial decrease occurs for larger aggregates. The latter two cases were experimentally observed and can be explained by the presence of larger off-diagonal anharmonicities for coupled N−H modes in hydrogen-bonded NEPA clusters. To summarize, our results revealed the influences of the solute−solute and solute−solvent interactions on the N−H stretching vibration in the model β-peptide, suggesting that the potential surface and vibrational dynamics of the N−H stretching mode is significantly altered when the NH group is involved in forming hydrogen-bonded peptide aggregates. Future work may include studying β-peptide structures and dynamics directly in water. It is, however, experimentally challenging because the O−H stretching vibration frequency of water overlaps with the N−H stretching frequency of peptides. To tackle this problem, one may use thin-layered (a few micrometers) sample solution so that bulk water O−H will have limited IR optical density.

ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (No. 21173231 and 91121020) and the Chinese Academy of Sciences (No. Y2201228) is acknowledged. The authors thank J. Shi and Y. Zhao for technical assistance.

■

REFERENCES

(1) Krimm, S.; Bandekar, J. Vibrational Spectroscopy and Conformation of Peptides, Polypeptides, and Proteins. Adv. Protein Chem. 1986, 38, 181−364. (2) Greve, C.; Preketes, N. K.; Costard, R.; Koeppe, B.; Fidder, H.; Nibbering, E. T. J.; Temps, F.; Mukamel, S.; Elsaesser, T. N−H Stretching Modes of Adenosine Monomer in Solution Studied by Ultrafast Nonlinear Infrared Spectroscopy and Ab Initio Calculations. J. Phys. Chem. A 2012, 116, 7636−7644. (3) De Marco, L.; Thämer, M.; Reppert, M.; Tokmakoff, A. Direct Observation of Intermolecular Interactions Mediated by Hydrogen Bonding. J. Chem. Phys. 2014, 141, 034502. (4) Shmilovits-Ofir, M.; Gerber, R. B. Proton Transfer and Dissociation of GlyLysH+ following O−H and N−H Stretching Mode Excitations: Dynamics Simulations. J. Am. Chem. Soc. 2011, 133, 16510−16517. (5) Dreyer, J. Unraveling the Structure of Hydrogen Bond Stretching Mode Infrared Absorption Bands: An Anharmonic Density Functional Theory Study on 7-Azaindole Dimers. J. Chem. Phys. 2007, 127, 054309. (6) Hadjieva, B.; Ilieva, S.; Cheshmedzhieva, D.; Galabov, B. Conformation of Some Biologically Active Aromatic Ureas. Spectrochim. Acta, Part A 2005, 61, 1321−1326. (7) Petersen, P. B.; Roberts, S. T.; Ramasesha, K.; Nocera, D. G.; Tokmakoff, A.; Ultrafast, N−H Vibrational Dynamics of Cyclic Doubly Hydrogen-Bonded Homo- and Heterodimers. J. Phys. Chem. B 2008, 112, 13167−13171. (8) Lee, S.-H.; Mirkin, N. G.; Krimm, S. A Quantitative Anharmonic Analysis of the Amide A Band in α-Helical Poly(L-alanine). Biopolymers 1999, 49, 195−207. (9) Fidder, H.; Yang, M.; Nibbering, E. T. J.; Elsaesser, T.; Röttger, K.; Temps, F. N−H Stretching Vibrations of Guanosine−Cytidine Base Pairs in Solution: Ultrafast Dynamics, Couplings, and Line Shapes. J. Phys. Chem. A 2013, 117, 845−854. (10) Gawinkowski, S.; Walewski, L.; Vdovin, A.; Slenczka, A.; Rols, S.; Johnson, M. R.; Lesyng, B.; Waluk, J. Vibrations and Hydrogen Bonding in Porphycene. Phys. Chem. Chem. Phys. 2012, 14, 5489− 5503. (11) Yang, M.; Szyc, Ł.; Röttger, K.; Fidder, H.; Nibbering, E. T. J.; Elsaesser, T.; Temps, F. Dynamics and Couplings of N−H Stretching Excitations of Guanosine−Cytidine Base Pairs in Solution. J. Phys. Chem. B 2011, 115, 5484−5492. (12) Szyc, Ł.; Yang, M.; Nibbering, E. T. J.; Elsaesser, T. Ultrafast Vibrational Dynamics and Local Interactions of Hydrated DNA. Angew. Chem., Int. Ed. 2010, 49, 3598−3610. (13) Chen, J.; Park, J.; Hochstrasser, R. M. Nonlinear-Infrared Responses of the N−H Mode of Pyrrole in Carbon Tetrachloride by Heterodyned and Stimulated Echoes. J. Phys. Chem. A 2003, 107, 10660−10666. (14) Ilieva, S.; Hadjieva, B.; Galabov, B. Theory Supplemented by Experiment. Electronic Effects on the Rotational Stability of the Amide Group in p-Substituted Acetanilides. J. Org. Chem. 2002, 67, 6210− 6215.

G


Article

The Journal of Physical Chemistry B (15) Galabov, B.; Ilieva, S.; Hadjieva, B.; Dudev, T. N−H Stretching Frequencies and the Conformation of Substituted Ureas: An Ab Initio MO Study. J. Mol. Struct. 1997, 407, 47−51. (16) Wynne, K.; Galli, C.; Hochstrasser, R. M. Femtosecond Intermolecular Vibrational Motion in Pyrrole. Chem. Phys. Lett. 1992, 193, 17−22. (17) Skrovanek, D. J.; Painter, P. C.; Coleman, M. M. Hydrogen Bonding in Polymers. 2. Infrared Temperature Studies of Nylon 11. Macromolecules 1986, 19, 699−705. (18) Muthusubramanian, P.; Raj, A. S. Internal Modes and Normal Coordinate Analysis of Sulphamic Acid. J. Mol. Struct. 1982, 84, 25− 37. (19) Beke, T.; Csizmadia, I. G.; Perczel, A. Theoretical Study on Tertiary Structural Elements of β-peptides: Nanotubes Formed from Parallel-Sheet-Derived Assemblies of β-Peptides. J. Am. Chem. Soc. 2006, 128, 5158−5167. (20) Karlsson, A. J.; Pomerantz, W. C.; Weisblum, B.; Gellman, S. H.; Palecek, S. P. Antifungal Activity from 14-Helical β-Peptides. J. Am. Chem. Soc. 2006, 128, 12630−12631. (21) Pomerantz, W. C.; Abbott, N. L.; Gellman, S. H. Lyotropic Liquid Crystals from Designed Helical β-Peptides. J. Am. Chem. Soc. 2006, 128, 8730−8731. (22) Wu, Y.-D.; Han, W.; Wang, D.-P.; Gao, Y.; Zhao, Y.-L. Theoretical Analysis of Secondary Structures of β-Peptides. Acc. Chem. Res. 2008, 41, 1418−1427. (23) Shi, J.; Zhao, J.; Yang, F.; Wang, J. Femtosecond TwoDimensional Infrared Spectroscopy of N-Ethypropionamide. Acta Phys. Chem. Sin. 2013, 29, 695−700. (24) Shi, J.; Wang, J. Interaction Between Metal Cation and Unnatural Peptide Backbone Mediated by Polarized Water Molecules: Study of Infrared Spectroscopy and Computations. J. Phys. Chem. B 2014, 118, 12336−12347. (25) Wang, J.; Zhao, J.; yang, F.; Shi, J. Structural Dynamics of NEthylpropionamide Clusters Examined by Nonlinear Infrared Spectroscopy. J. Chem. Phys. 2015, 143, 185102. (26) Ghosh, A.; Hochstrasser, R. M. A Peptide’s Perspective of Water Dynamics. Chem. Phys. 2011, 390, 1−13. (27) Hamm, P.; Lim, M.; Hochstrasser, R. M. Structure of the Amide I Band of Peptides Measured by Femtosecond Nonlinear-Infrared Spectroscopy. J. Phys. Chem. B 1998, 102, 6123−6138. (28) Hamm, P.; Woutersen, S.; Rueping, M. On the Thermal Stability of β-Peptides: A Two-Dimensional Vibrational Spectroscopy Study. Helv. Chim. Acta 2002, 85, 3883−3894. (29) Rubtsov, I. V.; Wang, J.; Hochstrasser, R. M. Dual Frequency 2D-IR of Peptide Amide-A and Amide-I Modes. J. Chem. Phys. 2003, 118, 7733−7736. (30) Barbour, L. W.; Hegadorn, M.; Asbury, J. B. Watching Electrons Move in Real Time: Ultrafast Infrared Spectroscopy of a Polymer Blend Photovoltaic Material. J. Am. Chem. Soc. 2007, 129, 15884− 15894. (31) Bian, H.; Wen, X.; Li, J.; Chen, H.; Han, S.; Sun, X.; Song, J.; Zhuang, W.; Zheng, J. Ion Clustering in Aqueous Solutions Probed with Vibrational Energy Transfer. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 4737−4742. (32) van Wilderen, L. J. G. W.; Kern-Michler, D.; MuellerWerkmeister, H. M.; Bredenbeck, J. Vibrational Dynamics and Solvatochromism of the Label SCN in Various Solvents and Hemoglobin by Time Dependent IR and 2D-IR Spectroscopy. Phys. Chem. Chem. Phys. 2014, 16, 19643−19653. (33) Asplund, M. C.; Zanni, M. T.; Hochstrasser, R. M. TwoDimensional Infrared Spectroscopy of Peptides by Phase-Controlled Femtosecond Vibrational Photon Echoes. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 8219−8224. (34) Mukherjee, P.; Kass, I.; Arkin, I.; Zanni, M. T. Picosecond Dynamics of a Membrane Protein Revealed by 2D IR. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3528−3533. (35) King, J. T.; Arthur, E. J.; Brooks, C. L., III; Kubarych, K. J. Crowding Induced Collective Hydration of Biological Macromolecules over Extended Distances. J. Am. Chem. Soc. 2014, 136, 188−194.

(36) Cahoon, J. F.; Sawyer, K. R.; Schlegel, J. P.; Harris, C. B. Determining Transition-State Geometries in Liquids Using 2D-IR. Science 2008, 319, 1820−1823. (37) Costard, R.; Tyborski, T.; Fingerhut, B. P.; Elsaesser, T. Ultrafast Phosphate Hydration Dynamics in Bulk H2O. J. Chem. Phys. 2015, 142, 212406. (38) Remorino, A.; Korendovych, I. V.; Wu, Y.; DeGrado, W. F.; Hochstrasser, R. M. Residue-Specific Vibrational Echoes Yield 3D Structures of a Transmembrane Helix Dimer. Science 2011, 332, 1206−1209. (39) Zheng, J.; Kwak, K.; Xie, J.; Fayer, M. D. Ultrafast CarbonCarbon Single-Bond Rotational Isomerization in Room-Temperature Solution. Science 2006, 313, 1951−1955. (40) Kolano, C.; Helbing, J.; Kozinski, M.; Sander, W.; Hamm, P. Watching Hydrogen-Bond Dynamics in a β-Turn by Transient TwoDimensional Infrared Spectroscopy. Nature 2006, 444, 469−472. (41) Fecko, C. J.; Eaves, J. D.; Loparo, J. J.; Tokmakoff, A.; Geissler, P. L. Ultrafast Hydrogen-Bond Dynamics in the Infrared Spectroscopy of Water. Science 2003, 301, 1698−1702. (42) Hamm, P.; Zanni, M. Concepts and Methods of 2D Infrared Spectroscopy; Cambridge University Press: 2011. (43) Cho, M. Two-Dimensional Optical Spectroscopy; CRC Press: 2009. (44) Fennell, C. J.; Bizjak, A.; Vlachy, V.; Dill, K. A. Ion Pairing in Molecular Simulations of Aqueous Alkali Halide Solutions. J. Phys. Chem. B 2009, 113, 6782−6791. (45) Lins, R. D.; Rö thlisberger, U. Influence of Long-Range Electrostatic Treatments on the Folding of the N-Terminal H4 Histone Tail Peptide. J. Chem. Theory Comput. 2006, 2, 246−250. (46) Norberg, J.; Nilsson, L. On the truncation of long-range electrostatic interactions in DNA. Biophys. J. 2000, 79, 1537−1553. (47) Darden, T. F. A. U. Y. D.; York, D. F. A. U. P. L.; Pedersen, L. Particle Mesh Ewald: An N*log(N) Method for Ewald Sums in Large Systems FAU - Darden Tom. J. Chem. Phys. 1993, 98, 10089. (48) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kalé, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781− 1802. (49) Jamróz, M. H. Vibrational Energy Distribution Analysis VEDA 4. Warsaw, 2004. (50) Wang, J.; Hochstrasser, R. M. Anharmonicity of Amide Modes. J. Phys. Chem. B 2006, 110, 3798−3807. (51) Zhao, J.; Wang, J. Understanding the Amide-II Vibrations in Beta-Peptides. J. Phys. Chem. B 2015, 119, 14831. (52) Jin, T.; Zhang, B.; Song, J.; Jiang, L.; Qiu, Y.; Zhuang, W. Infrared Signature of the Early Stage Microsolvation in the NaSO4− (H2O)1−5 Clusters: A Simulation Study. J. Phys. Chem. A 2014, 118, 9157−9162. (53) Moran, A.; Mukamel, S. The Origin of Vibrational Mode Couplings in Various Secondary Structural Motifs of Polypeptides. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 506−510. (54) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09, Revision A.02; Gaussian, Inc.: Pittsburgh, PA, 2009. (55) Lu, T.; Chenm, F. Multiwfn: A Multifunctional Wavefunction Analyzer. J. Comput. Chem. 2012, 33, 580−592. (56) Roberts, S. T.; Loparo, J. J.; Tokmakoff, A. Characterization of Spectral Diffusion from Two-Dimensional Line Shapes. J. Chem. Phys. 2006, 125, 084502. (57) Kwak, K.; Park, S.; Finkelstein, I. J.; Fayer, M. D. FrequencyFrequency Correlation Functions and Apodization in Two-Dimensional Infrared Vibrational Echo Spectroscopy: A New Approach. J. Chem. Phys. 2007, 127, 124503. (58) Kwak, K.; Rosenfeld, D. E.; Fayer, M. D. Taking Apart the TwoDimensional Infrared Vibrational Echo Spectra: More Information and Elimination of Distortions. J. Chem. Phys. 2008, 128, 204505. H


Article

The Journal of Physical Chemistry B (59) King, J. T.; Kubarych, K. J. Site-Specific Coupling of Hydration Water and Protein Flexibility Studied in Solution with Ultrafast 2D-IR Spectroscopy. J. Am. Chem. Soc. 2012, 134, 18705−18712. (60) Wang, J. Conformational Dependence of Anharmonic Vibrations in Peptides: Amide-I Modes in Model Dipeptide. J. Phys. Chem. B 2008, 112, 4790−4800. (61) Park, J.; Hochstrasser, R. M. Multidimensional Infrared Spectroscopy of a Peptide Intramolecular Hydrogen Bond. Chem. Phys. 2006, 323, 78−86. (62) Nitzan, A.; Mukamel, S.; Jortner, J. Energy Gap Law for Vibrational Relaxation of a Molecule in a Dense Medium. J. Chem. Phys. 1975, 63, 200−207.

I


Selectively Probing the Structures and Dynamics of Î²-Peptide

Recommend Documents