Conformations of Protonated AlaDap and DapAla Characterized by

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

pubs.acs.org/JPCB

Conformations of Protonated AlaDap and DapAla Characterized by IRMPD Spectroscopy and Molecular Modeling Patrick Batoon,† Jos Oomens,‡ Giel Berden,‡ and Jianhua Ren*,† †

Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, California 95211, United States FELIX Laboratory, Institute for Molecules and Materials, Radboud University, Toernooiveld 7c, 6525ED Nijmegen, The Netherlands

‡

S Supporting Information *

ABSTRACT: Oligopeptides containing 2,3-diaminopropionic acid (Dap) serve as a unique model to study conformational effects on the ionizability of a side-chain group. In this study, conformations of acetylated isomeric dipeptide ions containing alanine (Ala) and Dap, AlaDapH+ and DapAlaH+, are studied by infrared multiple photon dissociation (IRMPD) spectroscopy and computation. The IRMPD spectra are characterized in detail by comparing them with theoretical IR spectra of a set of low-energy conformations calculated at the ωB97X-D/6311+G(d) level of theory. The averaged IR spectra according to the Boltzmann distribution of the set of conformations have a good match to the IRMPD spectra. The characteristic amide I band of AlaDapH+ appears to be downshifted compared to that of DapAlaH+. The relative positions of the amide band suggest a stronger hydrogen-bonding interaction between the charged side-chain amino group and the amide carbonyl groups in AlaDapH+ than in DapAlaH+. The stronger hydrogen bonding in the former is likely due to a better alignment of the N−H and OC bonds, which enables an effective sequestering of the positive charge at the amino group. The effect results in a higher proton affinity of acetylated dipeptides with the Dap residue at the C-terminus.

1. INTRODUCTION The compound 2,3-diaminopropionic acid (Dap) is a nonproteinogenic amino acid produced by numerous plants and bacteria.1 Dap functions as a key precursor in the biosynthesis of antibiotics in plants and bacteria.2 N-terminal Dapcontaining dipeptides have been found to serve as efficient methylglyoxal scavengers to inhibit advanced glycation endproduct formation.3,4 Chemically synthesized Dap-based compounds have been used as an efficient gene-delivery system.5 Dap is a lysine (Lys) homologue containing an ionizable side-chain amino group. For many proteins, the active sites with residues containing an ionizable side-chain group are found inside the hydrophobic core, which is formed by hydrophobic residues arranged in such a way that solvent interactions are minimized.6 Residues with ionizable side-chain groups existing within the solvent-free core have shown to play important roles as reactive agents that carry out protein function.7 Their reactivity is largely connected to the acid−base property, or the pKa values, of the residues in the active sites. The effective pKa values of the buried residues with ionizable groups, such as lysine (Lys), are highly dependent on the conformations of the microenvironment within the active sites.8 In this regard, peptides containing Lys and Lys homologues can be used as a model to emulate different microenvironments with a basic residue buried inside a protein. Studies of the gas© XXXX American Chemical Society

phase proton affinity and basicity of different model peptides will greatly help to understand the biochemistry inside proteins. In the past two decades, there have been substantial studies on the proton affinity and gas-phase basicity of various peptides as well as amino acids.9−22 Like other peptides consisting of nonproteinogenic Lys homologues, Dap-containing oligopeptides can serve as a unique model to examine the effect of the backbone conformation and the ionizability of the side-chain group. Compared to Lys, Dap has a much shorter side chain with only one methylene group (CH2) separating the ionizable amino group and the backbone. Therefore, Dap-containing peptides are expected to have reduced conformational flexibility. We have recently studied a series of N-terminal acetylated dipeptides containing alanine (Ala) and a basic residue containing a side-chain amino group, such as Lys and Lys homologues, including Dap.23,24 The peptides are divided into two sets, one with the basic residue (X) at the C-terminus (AlaX) and the other at the N-terminus (XAla) (Scheme 1). The two sets display a notable difference in terms of proton affinity. The peptides having a C-terminal basic residue appear Received: October 21, 2017 Revised: January 28, 2018 Published: January 29, 2018 A

DOI: 10.1021/acs.jpcb.7b10435 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Scheme 1. Structures of Acetylated Dipeptides with an Ionizable Amino Group

Scheme 2. Structures of Acetylated AlaDapH+ and DapAlaH+

photon dissociation (IRMPD) experiments were carried out at FELIX Laboratory (Radboud University Nijmegen, The Netherlands) using a 4.7 T Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer coupled to the FELIX infrared free-electron laser. The principles and the procedure of the IRMPD experiments have been described previously by Eyler, Oomens, Polfer, and their co-workers.25,30,59−61 To perform the IRMPD experiment, peptide solutions were prepared by dissolving the solid form of the peptide into a 1:1 (v/v) mixture of acetonitrile/water to achieve a concentration of about 0.5 mM. The sample solution was infused into the electrospray ionization (ESI) source at a flow rate of 5−10 μL min−1. The protonated peptide ions were generated in the ESI source with nitrogen drying gas at about 95 °C to facilitate desolvation. The peptide ions were accumulated for 3 s in an radio frequency hexapole ion trap and then pulsed into the ICR cell. Unwanted ions were ejected by a stored waveform inverse Fourier transform excitation pulse. The isolated peptide ions were then irradiated by 12 infrared laser pulses of the FELIX free-electron laser tuned to frequencies in the range of 500−1800 cm−1. The infrared beam arrives as 5 μs long macropulses with a bandwidth of 0.4% of the center frequency. Pulse energies in the range of 15−60 mJ were used to dissociate the ions. Upon resonance between a vibrational band of the peptide ion and the laser frequency, absorption of multiple infrared photons occurs, resulting in internal heating of the trapped ions up to a dissociation threshold via rapid intramolecular vibrational redistribution.60,25,59 The energized ions undergo unimolecular dissociation to produce corresponding fragment ions. The intensities of mass peaks due to the peptide ion and its fragment ions are recorded in the FT-ICR mass spectrometer as the laser frequency is scanned across the desired IR spectral range. The fragmentation yield at a given frequency is calculated from the measured ion intensities using eq 1, where Iprecursor and Ifragments are the ion intensities of the precursor peptide ions and fragment ions, respectively. The IRMPD spectrum is obtained by plotting the IRMPD yield against the photon energy in wavenumbers (cm−1). The yield at each IR point is obtained from three averaged mass spectra and is linearly corrected for frequency-dependent variations in the laser power.30,49

to have a higher proton affinity than their counterparts with an N-terminal basic residue. For example, the proton affinities of AlaLys and LysAla have been determined to be 246.3 and 241.5 kcal mol−1, respectively. Similarly, the proton affinities of AlaDap and DapAla have been determined to be 237.0 and 234.5 kcal mol−1, respectively, with an increase of 2.5 kcal mol−1 by moving Dap from the N- to the C-terminus. Presumably, peptides with higher proton affinity adopt a conformation upon protonation, in which the charge can be better stabilized via internal solvation, such as hydrogenbonding interactions. However, in examining the computed lowest-energy conformations of all of the protonated peptides, no correlation between the hydrogen-bond distances can be found to indicate a difference in proton affinity. Instead, our computational studies suggest that the set of peptides with a Cterminal basic residue adopts a more compact conformation upon protonation. It will be interesting to probe the conformational differences between the two sets of the protonated peptides experimentally. Vibrational spectroscopy, especially infrared multiple photon dissociation (IRMPD) techniques, have been widely used to study the conformations of charged amino acids and oligopeptides.25−54 Specific examples of molecular systems include protonated, deprotonated, and metal cation adducts of amino acids;46,50−53,55−57 protonated and metal cation adduct of peptides;26,34−39,41,47 and fragments from and secondary structures of charged peptides.28,30,31,33,40,44,45,58 In this work, we focus on characterizing the conformations of the two protonated isomeric Dap-alanine peptides with N-terminal acetylation, AlaDapH+ and DapAlaH+, using a combination of IRMPD spectroscopy and computational modeling. The structures of the peptides are shown in Scheme 2, where Ac represents the acetyl group, and Dap and Ala represent the Dap residue and Ala residues, respectively. The N-terminal acetylation eliminates the possibility of protonation at the Nterminal amino group so that the only likely protonation site is the side-chain amino group.

2. EXPERIMENTAL AND COMPUTATIONAL METHODS 2.1. Experimental Methods. The peptides were synthesized via solid-phase peptide synthesis using a protocol described in our previous publications.23,24 Infrared multiple B


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The Journal of Physical Chemistry B IRMPD yield =

single-point energy (ε0) and the ωB97X-D thermal correction to free energy at 298 K (Gcorr) for each conformation, for which the thermal correction to zero-point energy was included. The final set of the conformations was ranked according to the values of the free energy and the Boltzmann probability (pi) calculated using eq 2, where εi represents the Gibbs free energy relative to the lowest-energy structure, R is the fundamental gas constant (1.987 × 10−3 kcal mol−1 K−1), and T is the temperature (298 K). For comparison, the Boltzmann probability was also calculated using the Gibbs free energies obtained from the frequency calculations at the ωB97X-D/6311+G(d) level.

∑ (Ifragments)

(Iprecursor + ∑ Ifragments)

(1)

2.2. Computational Methods. Conformations and energetics of protonated AlaDap and DapAla were calculated using a funneling approach for which a large number of conformations were first generated using a low-cost computational method and then funneled into successive calculations utilizing progressively higher levels of theory. The first step involved the generation of 10 000 conformers by Merck molecular force field (MMFF) using the Spartan ’08 suite (Wavefunction, Irvine, CA).62 For the pool of conformations generated using the MMFF, 100 of the most stable conformations were kept and subjected to AM1 optimization, and this was followed by geometry and frequency calculations at the HF/3-21G level. To obtain a more accurate energy ladder, the resulting 100 conformations were then subjected to optimization and frequency calculations with ωB97X-D functional using the 6-31+G(d) basis set. Degenerate structures were removed if two or more structures converged to the same geometry and energy. The final 10 lowest-energy conformations further underwent geometry optimization and frequency calculation at the ωB97X-D/6-311+G(d) level, and degenerate structures were removed. Finally, single-point energy calculations were carried out on the set of final structures at the MP2/6-311++G(2d,p) level of theory. All quantum-level calculations were carried out using the Gaussian 09 suite of program.63 The ωB97X-D functional for geometry optimizations was selected due to known deficiencies with the popular B3LYP method in dealing with dispersion interactions (London forces).64 Ion spectroscopy studies carried out with small oligopeptides and other biologically relevant molecules demonstrate the importance of employing dispersion-corrected density functional theory in dealing with hydrogen-bonding interactions involving π-electron-rich regions, such as in the case of a charged group with an aromatic ring.65−67 The ωB97X-D was developed by the Head-Gordon group and based on the older B97D functional.68 Compared to other dispersion and long-range corrected functionals, ωB97X-D was shown to have one of the lowest mean average differences for the S22 benchmark set of compounds69 and also shown to accurately predict proton affinities of small amine molecules.70 Although the peptides AlaDap and DapAla do not contain any aromatic residues, the choice of ωB97X-D takes into consideration the possible interaction between the charged site and the π-electron-rich peptide backbone. In fact, the B3LYP/6-311+G(d) method was applied to calculate the geometries and frequencies for the two peptide ions. When matching the calculated IR spectra with the IRMPD spectra, the results from the B3LYP functional were not as good as those obtained from ωB97X-D functional, especially in the region of amide bands. Upon geometry optimization of the selected conformations using the ωB97X-D functional, the angles and atomic distances associated with hydrogen-bonding interactions between NH3+ and OC and the values of the dipole moment are collected from each output file. The assessable surface area71 was calculated using Spartan software from the optimized conformations. After the MP2 single-point calculations, Gibbs free-energy values (ΔG298, 298 K) were obtained by summing the MP2

pi =

exp( −εi /RT ) ∑ exp( −εi /RT )

(2)

A uniform linear scaling factor of 0.950 was applied to the theoretical IR frequencies obtained from the ωB97X-D calculations, and the spectra were convoluted with a 30 cm−1 full width at half-maximum (FWHM) Gaussian profile to mimic the peak widths of the experimental spectra and to facilitate spectral matching with the experiment. In addition, a weighted average spectrum based on the spectra of the set of lowest-energy conformations for each peptide was created using the Boltzmann distribution. To characterize the bands in the IRMPD spectrum, the calculated IR vibrations associated with each conformation were compared to the frequencies of the IRMPD bands. The best-matched vibrations were visualized using the animation feature in GaussView 5 software,72 and the corresponding vibrational modes were assigned. For each charged peptide, various input structures with protonation sites at different nitrogen and oxygen atoms as well as at the side-chain amino group of Dap were considered. However, all resulting optimized structures either had converged into the side-chain-protonated Dap or had energetic values far greater than the lowest-energy structure; thus, only protonation at the side chain of Dap was considered in the initial input structure.

3. RESULTS 3.1. IRMPD Spectra of AlaDapH+ and DapAlaH+. Experimental IRMPD spectra of the two isomeric peptide ions, AlaDapH+ and DapAlaH+, are shown in Figure 1 with the major identifiable vibrational bands labeled. The shapes of the two spectra are apparently different, reflective of the unique fine structures of the peptide ions. One major difference is in the shifted positions of characteristic peptide vibrations, amide I (commonly at ∼1650 cm−1) and amide II (commonly at ∼1515 cm−1). For the spectrum of AlaDapH+ (Figure 1a), the two bands, amide I (1637 cm−1) and amide II (1503 cm−1), are apparently downshifted and the frequency difference between these two bands (134 cm−1) is relatively small. On the other hand, for the spectrum of DapAlaH+ (Figure 1b), both amide I (1685 cm−1) and amide II (1520 cm−1) are upshifted and the frequency difference (165 cm−1) is relatively large. Another major difference is the degree of broadening of the spectral peaks; the bands for DapAlaH+ appear to have a higher degree of broadening. 3.2. Calculated Conformations of AlaDapH+ and DapAlaH+. Conformations and relative energies of protonated AlaDap and DapAla were calculated using a funneling approach. The final geometry was optimized at the ωB97XD/6-311+G(d) level of theory and the energy was calculated C


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ions, but the angle of H···OC is clearly larger for AlaDapH+ than for DapAlaH+. The free energies and the corresponding Boltzmann distributions calculated at the ωB97X-D/6311+G(d) and MP2/6-311++G(2d,p)//ωB97X-D/6311+G(d) levels of theory are shown in Table 1. Results from both levels of calculation suggest that the most likely conformations for AlaDapH+ are A and B, and for DapAlaH+ are U and V. 3.3. Calculated IR Spectra of AlaDapH+ and DapAlaH+. The calculated IR spectra for the selected conformations are shown in Figure 3 for AlaDapH+ and in Figure 4 for DapAlaH+ along with the IRMPD spectrum (in black) recorded for each of the two systems. As the IR spectra of the lowest-energy conformations, A and B for AlaDapH+ and U for DapAlaH+, present a good overall match with the corresponding IRMPD spectrum, the IR spectra of other conformations also show a reasonable match with the IRMPD spectra. In fact, in a direct comparison of the IRMPD spectra to the calculated IR spectrum for each conformation, it appears that C and D most closely resemble the IRMPD spectrum of AlaDapH+, whereas V and X most closely represent the IRMPD spectrum of DapAlaH+. Each experimental IRMPD spectrum likely represents a thermally averaged population of conformations. To investigate this possibility, the Boltzmann-averaged spectra for each peptide ion calculated at both levels of theory of all selected conformations are generated. Although similar, the averaged spectra resulting from ωB97X-D/6-311+G(d) calculations (Figure 5) have slightly better matching with the IRMPD spectra than the ones resulting from MP2/6-311+ +G(2d,p)//ωB97X-D/6-311+G(d) calculations (Figure S2), especially at the amide I region of AlaDapH+. Compared to all individual spectra, the averaged spectra produce the best overall match with the IRMPD spectra. 3.4. Spectral Data Assignment. 3.4.1. IRMPD Assignment for AlaDapH+. The assignment of the bands in the experimental IRMPD spectrum of AlaDapH+ (Figure 1a) is shown in Table 2, in which the vibrational modes of the bestmatched conformations are compared to the characteristic peaks in the IRMPD spectrum. The nomenclature used is as follows: ν represents stretching motions between two atoms, δ represents in-plane bending motions (usually between three atoms), and γ represents out-of-plane bending motions. The highest-energy vibrations at 1782 and 1751 cm−1 are primarily due to the characteristic CO stretching of the C-terminal carboxyl group. The splitting of the peak indicates contributions from different conformations. The peak at 1782 cm−1 relates to conformations B and D, for which the CO stretch is coupled to the bending mode of the C-terminal OH and the Dap amide CN−H. The peak at 1751 cm−1 comes from conformation F, for which the CO stretching is coupled to a NH3+ pendulum motion. The group of bands centered at 1637 cm−1 is assigned as the amide I vibration. The amide I vibration (normally at ∼1650 cm−1) arises mainly from the stretching of the amide CO bonds and having minor contributions from the C−N out-ofphase stretching and the N−H in-plane bending.73,74 The amide I vibration is widely utilized as a diagnostic of the degree and type of hydrogen bonding and is commonly used to determine the secondary structure of peptides and proteins. The first related vibration in the amide I system is the shoulder peak at 1692 cm−1 originating from Ala CO stretching, CN− H bending of the Dap amide, and bending around the Ala αcarbon. The NH3+ pendulum motion is highly coupled to the

Figure 1. Experimental IRMPD spectra of AlaDapH+ and DapAlaH+ recorded in the IR range of 500−1800 cm−1, where the values in red indicate the estimated centers of the vibrational bands.

using the MP2/6-311++G(2d,p)//ωB97X-D/6-311+G(d) procedure. The resulting seven unique conformations for AlaDapH+ (A−G) and six unique conformations for DapAlaH+ (U−Z) are summarized in Figure 2. Detailed geometric information is shown in Figure S1 (Supporting Information). The likely hydrogen-bonding interactions between the charged NH3+ group and the oxygen atom of the carbonyl groups are represented by dashed lines along with values for the “bond lengths” in angstroms (Å). An upper limit of 2.6 Å between the hydrogen and oxygen atoms is used to assign a possible hydrogen-bonding interaction. Most of the values are in the range of 1.5−2.5 Å. The conformations of AlaDapH+ display up to four possible hydrogen-bonding interactions between the NH3+ group and oxygen atoms. For example, conformation A shows possible interactions of NH3+···OAc, NH3+···OAla, and NH3+···ODap. The conformations of DapAlaH+ display two possible hydrogen-bonding interactions. For example, conformation U shows interactions of NH3+···OAc and NH3+···OAla. Geometric and other molecular properties calculated at the ωB97X-D/6-311+G(d) level of theory are summarized in Table 1. These properties include angles associated with hydrogen-bonding interactions (N−H···O and H···OC), the dipole moment, and the accessible surface area. To help evaluate the trends in these values, the average values for each of the parameters are listed in Table 1 as well. Overall, the dipole moment and the accessible surface area for the conformations of AlaDapH+ are smaller than those for the various low-energy conformations of DapAlaH+. With respect to the angles associated with hydrogen-bonding interactions, the values for N−H···O are comparable for the two peptide D


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Figure 2. Lowest-energy conformations of AlaDapH+ (A−G) and DapAlaH+ (U and V) calculated at the ωB97X-D/6-311+G(d) level of theory. The dashed lines represent likely hydrogen-bonding interactions accompanied by bond lengths in angstroms (Å), for which 2.6 Å is the upper limit.

The next set of peaks centered at 1385 and 1268 cm−1 is in the range of the commonly known amide III vibrations (normally between 1400 and 1200 cm−1) caused by the combination of amide N−H bending, C−N stretching, CO bending, as well as C−C stretching.73,74 The peak at 1385 cm−1 is a due to various vibrational modes, including stretching of the C−N bonds, bending of the CN−H bonds, bending around the α-carbon of Dap, and bending and stretching of the C-terminal COOH group. This peak is best matched by conformations B, C, and D, with slight upshifts and downshifts. The shifts are likely contributing to the observed broadening of the IRMPD band. The smaller peak at 1268 cm−1 can be attributed to C−N stretching and CN−H bending coupled to the bending of the Cα−H bonds of both residues. The conformations best matching to this peak are A and D. A pair of peaks at 1158 and 1122 cm−1 is not characterized as “amide” vibrations because they are not associated with any significant vibrations in the amide backbone. Several conformations appear to have bands around this region, in particular conformations B, C, and D. The last pair of peaks observed at 638 and 582 cm−1 may be classified under the amide IV, V, and VI vibrations (normally in the range of 530− 770 cm−1).74 But they cannot be fully assigned due to their low resolution. Conformations B, D, and G appear to have weak IR bands at this region. 3.4.2. IRMPD Assignment for DapAlaH+. The assignment of the bands in the IRMPD spectrum of DapAlaH+ (Figure 1b) is

Ala CO stretching, which is likely due to strong hydrogen bonding. The peak at 1692 cm−1 has a good match with the IR spectra of conformations B, D, and G. The larger, more intense peak at 1637 cm−1 of the amide I system is a result of the same vibrations but intense motions of the CO stretching coupled to the NH3+ pendulum and scissoring motions. The best match to this band is with the IR spectrum of conformation C. Inclusion of the NH3+ motions in the amide I bands indicates protonation solely at the side-chain site as other vibrational modes related to protonated COH+ are not observed. The next major group of bands centered at 1503 cm−1 is assigned to the vibrations of the amide II system. The amide II vibration (normally at ∼1550 cm−1) results from the amide N− H in-plane bending and C−N stretching.73,74 The small highfrequency shoulder at 1555 cm−1 is mainly due to the strong bending motion of the amide N−H and C−N stretchings of Dap, and the strong NH3+ pendulum and scissor motion coupled to slight stretching of the CO group of Ala is due to hydrogen bonding. The best match to this peak comes from the IR spectrum of conformation F. The major peak at 1503 cm−1 is due to the bending motion of the amide N−H bond at the AlaDap backbone and the bending motion of the Cα−H of both Ala and Dap residues. The band at 1503 cm−1 is predicted in all IR spectra of the group of selected conformations and best matching with experiment for the IR spectra of A, D, and E. E


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Table 1. Summary of Structural Features, Molecular Properties, and Relative Energies for the Lowest-Energy Conformations of AlaDapH+ and DapAlaH+ ΔG298 (kcal mol−1)

angles (deg)a conf.

N−H···O

H···OC

dipole (D)

acc. area (Å )

ωB97XD

MP2

160.8 170.9 174.3 173.7 167.0 153.2 173.7 167.1

149.0 138.1 153.0 146.2 147.8 121.3 146.6 141.7

4.74 4.02 3.36 3.57 5.64 5.69 3.07 4.23

144.2 151.0 148.9 149.2 144.6 152.0 148.6 149.1

0.43 0.00 0.69 1.41 2.79 0.97 2.86

178.0 147.9 172.7 147.3 151.4 147.6 168.6

134.6 125.3 133.0 125.4 124.2 126.3 131.7

5.45 7.15 5.05 9.87 4.54 9.42 5.92

148.5 154.0 148.1 154.8 152.4 148.1 150.1

0.00 0.43 1.13 4.00 4.32 6.32

a

2 a

b

Boltzmann distribution (pi), % ωB97XDd

MP2e

0.00 0.50 1.41 1.79 2.09 2.11 3.19

22.99 47.65 14.88 4.43 0.43 9.25 0.38

61.23 26.34 5.61 3.00 1.80 1.74 0.28

0.00 0.06 1.02 3.39 4.82 5.04

61.26 29.58 9.05 0.04 0.07 0.001

48.00 43.28 8.54 0.16 0.014 0.010

c

+

AlaDapH A B C D E F G avgf DapAlaH+ U V W X Y Z avgf a

Angles associated with hydrogen-bonding interactions (N−H···O and H···OC), dipole moment, and accessible surface area are calculated at the ωB97X-D/6-311+G(d) level of theory. We note that the hydrogen-bonding interactions refer to the bonds with the shortest H···O distance. bValues of ΔG298 are calculated at the ωB97X-D/6-311+G(d) level of theory. cValues of ΔG298 are calculated at the MP2/6-311++G(2d.p)//ωB97X-D/6311+G(d) level of theory. dValues of Boltzmann distribution (pi) are obtained using the values of ΔG298 calculated at the ωB97X-D/6-311+G(d) level of theory. eValues of Boltzmann distribution (pi) are obtained using the values of ΔG298 calculated at the MP2/6-311++G(2d.p)//ωB97X-D/6311+G(d) level of theory. fValues of weighted average are calculated using the Boltzmann distribution obtained from ωB97X-D/6-311+G(d).

Figure 4. Computed IR spectra of the six conformations of DapAlaH+ (bold blue line) overlaid onto the experimental IRMPD spectrum (fine black line), where the values indicate the relative free energies (Table 1) calculated at the ωB97X-D/6-311+G(d) (in parenthesis) and MP2/6-311+G(2d,p)//ωB97X-D/6-311+G(d) levels of theory, respectively.

Figure 3. Computed IR spectra for the seven conformations of AlaDapH+ (bold red line) overlaid onto the experimental IRMPD spectrum (fine black line), where the values indicate the relative free energies (Table 1) calculated at the ωB97X-D/6-311+G(d) (in parenthesis) and MP2/6-311+G(2d,p)//ωB97X-D/6-311+G(d) levels of theory, respectively.

terminal CO stretching and the amide I vibrations. The intense peak at 1759 cm−1 can be attributed to stretching of the C-terminal CO bond and bending of the C−O−H group, which is clearly shown in conformation V. The next peak observed is sandwiched within the cluster at 1736 cm−1 and is a combination of CO stretching of the C-terminal carboxyl

shown in Table 3. The first cluster of peaks appearing from 1759 to 1685 cm−1 are an overlapping mixture of the CF


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The most intense peak at 1520 cm−1 is representative of the characteristic amide II vibration, which is due to strong CN−H bending motion in the amide linkages between the acetyl and Dap residues as well as between the Dap and Ala residues, and which matches well with the IR vibrations of the conformations of W, X, and V. The amide-bending motion is accompanied by NH3+ umbrella motion (clearly seen in W), presumably due to through-bond coupling. The peak at 1459 cm−1 in DapAlaH+ resembles the one at 1454 cm−1 in AlaDapH+, but has a much larger intensity. As shown from the vibrations of the closest-matching conformations (V, X, Z), this peak is likely due to the strong umbrella motion of NH3+ coupled to the scissoring of the CH2 of the Dap side chain. The peak at 1335 cm−1 matches with the vibrations of conformation Y. The peak at 1263 cm−1 is assigned to the amide III vibration. Conformation U has a weak IR band matching well to this peak. This amide III peak is mainly due to the bending of the CN−H group of Ala coupled with a strong out-of-plane bending of the Cα−H group of both residues. The next broad band with two maxima at 1159 and 1121 cm−1 arises from a combination of motions and is not characterized as “amide” vibrations. Conformations U and X have vibrations matching the peak at 1159 cm−1. Conformation V has an IR band matching the peak at 1121 cm−1. The next two peaks centered at 879 and 737 cm−1 match well with the vibrations of conformation V. The peak at 879 cm−1 is unique to DapAlaH+ and is apparently due to intense out-of-plane bending motions of CH2 and NH3+ groups of Dap along with

Figure 5. Boltzmann-averaged IR spectra for the two protonated dipeptides AlaDapH+ (red) and DapAlaH+ (blue) compared with the experimental IRMPD spectra (black). The Boltzmann distribution is based on the ΔG298 values calculated at the ωB97X-D/6-311+G(d) level of theory.

group, the Dap CO group, and the acetyl CO group (conformation U). Hydrogen-bonding interaction between the charged NH3+ group and the nearby CO groups causes a slight scissoring motion of the NH3+ group. The most intense peak of this cluster at 1685 cm−1 is a characteristic amide I vibration, which is attributed to the strong CO stretching of Dap coupled to weak stretching of the C−N bond. A strong pendulum and scissoring motion of the charged NH3+ group is likely due to the strong hydrogen-bonding interaction with the Dap CO group. Three conformations V, Z, and Y all have an IR vibration matching well with this peak.

Table 2. IRMPD Spectral Peaks with Approximate Mode Descriptions and Frequencies Calculated for AlaDapH+ obs.a 1782 1751 1692

1637 1555 1503

1385

1268 1158 1122 638

582

calcdb 1787 1785 1747 1697 1694 1702 1631 1550 1512 1502 1499 1498 1489 1370 1400 1359 1260 1258 1169 1157 1137 633 632 635 575 564

conf.c B D F B D G C F D A E A D B C D A D C B D B D G D G

vibrational modesd νCO

amide I

amide II

amide III

amide IV, V, VI

Dap

, δC−O−H

Dap

, δCN−HDap (minor)

νCODap, δC−O−HDap, δNH3+ (pendulum) νCOAla, δCN−HDap, δNH3+ (pendulum, scissor), δCα−HAla (minor)

νCOAc, δCN−HAla, δNH3+ (scissor) δCN−HDap, δNH3+ (pendulum, scissor), νC−NHAla−Dap δCN−HAla, νC−NHAc−Ala, δCα−HAla δCN−HDap, γNH3+ (umbrella), δCα−HDap δCN−HAla, νC−NHAc−Ala δCN−HAla, νC−NHAc−Ala, γNH3+ (umbrella), δCα−HAla δCN−HDap, νC−NHAla−Dap, δCα−HDap νC−NHAla−Dap, δCN−HDap, νCα−COAla, δNCα−HAla, γCH3Ala (umbrella) νC−NHAla−Dap, δCN−HDap, δCα−HDap, νCα−CODap, νC−OHDap, δC−O−HDap, γCH2Dap νC−NHAla−Dap, δCα−HDap, νC−OHDap, δC−O−HDap, γCH3Ac νC−NHAc−Ala, δCN−HAla, νC−NHAla−Dap, δCN−HDap, γCα−HAla, γCα−HDap, δC−O−HDap νC−NHAc−Ala, δCN−HAla, γCα−HAla, γCH3Ac (umbrella) δC−O−HDap, δCN−HDap, γCH2Dap (rocking) δC−O−H, νN−Cα,Ala, δCN−HDap δC−O−H, νN−Cα,Dap, γNH3+ (rocking) γO−H, δOC−OH, γOC−NAc−Ala γO−H, δOC−OH, γOC−NAc−Ala, γC−N−HAla γO−H, γOC−OH, γCN−HAla, γC−N−HDap

Peak positions taken from the experimental IRMPD spectrum. bCalculated IR frequencies at the ωB97X-D/6-311+G(d) level and scaled by 0.95. Letters corresponding to the conformations shown in Figure 1. dν represents stretching motion, δ represents in-plane bending motion, and γ represents out-of-plane bending motion. a c

G


Article

The Journal of Physical Chemistry B Table 3. IRMPD Spectral Peaks and Best-Matched IR Frequencies Calculated for DapAlaH+ obs.a 1759 1736 1685

calcdb

1121 879 737 612

1755 1738 1686 1689 1692 1519 1523 1518 1521 1459 1461 1457 1342 1269 1166 1159 1122 867 731 616

583

585

1520

1459

1335 1263 1159

conf.c V U V Z Y W W X V V X Z Y U U X V V V V Y V

vibrational modesd νCO , δC−O−H, δC−N−HAla, δN−Cα−HAla νCOAla, νCODap, νCOAc, δC−O−H, δC−N−HAla, νC−NDap−Ala, δNH3+ (scissor) νCODap, δNH3+ (pendulum), νC−NDap−Ala νCODap, δNH3+ (scissor), νC−NDap−Ala, νCOAc νCODap, δNH3+ (scissor), νC−NDap−Ala, νCOAc δCN−HAla, γNH3+ (umbrella) δCN−HAc, γNH3+ (umbrella) γCN−HAla, δCN−HDap γCN−HAla, δCN−HDap, νC−NDap−Ala γNH3+ (umbrella), δCH2 (scissor) γNH3+ (umbrella), δCH2 (scissor) γNH3+ (umbrella), δCH2 (scissor) δC−O−H, γCH3Ala, νCα−COAla, γCH2, νCα−CODap δCN−HAla, γCα−HDap, γCα−HAla, γNH3+ (rocking), δC−O−H δC−O−H, νCα−NAla, νCα−NDap, γCH2, γNH3+ (rocking) δC−O−H, νC−OH, νCα−NAla, γCH3, γNH3+ (rocking) δC−O−H, νCα−CNAla, γCH3 (rocking), δNH3+ (rocking), δCH2, νCα−CODap γCH2Dap, γNH3+ (rocking), δOC−NAla, γCH3Ala (rocking) γOC−OH, γO−H, γCα−COAla γO−H, γCN−HAla, γOC−N Ala

amide I

amide II

amide III

amide IV, V, VI

γCO−H, γCN−HAla

Peak positions taken from the experimental IRMPD spectrum. bCalculated IR frequencies at the ωB97X-D/6-311+G(d) level and scaled by 0.95. Letters corresponding to the conformations shown in Figure 1. dν represents stretching motion, δ represents in-plane bending motion, and γ represents out-of-plane bending motion. a c

calculated IR vibrational modes, and comparable vibrational modes are assigned to both IRMPD spectra. 4.2. Characteristics of the IRMPD Spectra. The IRMPD spectra of the two isomeric peptide ions are clearly different, particularly in the regions of the amide I and amide II vibrations. For proteins and peptides, in general, the overall secondary structure can be determined using these two characteristic bands.73,74 The amide I (predominantly CO stretching) and amide II (predominantly CN−H bending and C−N stretching) bands have been shown to be sensitive to hydrogen-bonding interactions.75 Hydrogen bonding at the carbonyl group would stabilize the charged resonance structure of the amide bond, which results in an increase in the CN double-bond character as the CO bond order decreases. The amide I peak would be downshifted due to truncation of CO stretching, and the amide II peak would be upshifted due to enhanced C−N bond order. As a result, the frequency differences are smaller for the shifted bands.75 As shown in Figure 1, both of the amide I and II bands of AlaDapH+ (1637 and 1503 cm−1) appear to be downshifted compared to the two bands of DapAlaH+ (1685 and 1520 cm−1). However, the frequency difference between amide I and amide II is smaller for AlaDapH+ (Δṽ = 134 cm−1) than for DapAlaH+ (Δṽ = 165 cm−1). On the basis of the resonance model and the relationship between hydrogen-bonding interactions and shifts in the amide IR bands, one can predict that AlaDapH+ is more strongly hydrogen-bonded than DapAlaH+ at the amide CO groups. Although both of the IRMPD spectra show relatively broad bands, the bands for DapAlaH+ appear to have a slightly higher degree of broadening. As a general rule of thumb, wider bands are indicative of a greater number of degrees of flexibility and an increased number of conformational contributions.73 The broadened peak widths in the IRMPD spectra, for example at

bending motions of the OC−N amide group (in-plane bending) and side-chain CH3 of Ala. The final two peaks at 612 and 583 cm−1 are in the range of amide IV, V, and VI vibrations. They are mainly due to out-ofplane bending motions of O−H of the C-terminus and CN−H of Ala and puckering of the OC−N groups. Conformations V and Y have weak IR bands matching these two peaks.

4. DISCUSSION 4.1. Summary of Results. The IRMPD spectra of the two isomeric dipeptides are characteristically different (Figure 1). One major difference is the positions of the amide I and amide II bands. For AlaDapH+, the two bands appear to be downshifted (red-shifted) and are relatively closer in frequencies, whereas for DapAlaH+, the two bands are upshifted (blue-shifted) and are more separated in frequencies. The second difference is that the bands in the spectrum of DapAlaH+ are relatively broader. Molecular modeling yields seven unique lowest-energy conformations for AlaDapH+ within 3 kcal mol−1 and six unique conformations for DapAlaH+ within 5 kcal mol−1 (Figure 2). Regarding the structural properties of the two peptide ions (Table 1), the angles associated with hydrogen bonding (H2N+−H···OC) are larger for AlaDapH+ than for DapAlaH+, and both the dipole moment and the accessible surface area (small difference though) are smaller for the former. When matching the calculated IR spectra of the selected conformations with the IRMPD spectra, although the ones of the lowest-energy conformations have an overall good match, several other conformations also have characteristic bands matching with the IRMPD spectra (Figures 3 and 4). In fact, the Boltzmannaveraged IR spectra provide the best match with the experimental IRMPD bands (Figure 5). All observable bands in the IRMPD spectra are characterized according to the H


Article

The Journal of Physical Chemistry B the amide I and amide II regions (with a resolution of ∼30 cm−1 FWHM), can be rationalized as the overlapping contributions of multiple low-energy conformations. For the AlaDapH+ system shown in Figure 3, one lowest-energy conformation, A, overestimates amide I but has a good match with the peak at amide II. In contrast, another lowest-energy conformation, B, has a good prediction for amide I band but overestimates amide II. The slightly higher-energy conformations, C and D, appear to have a good prediction of the overall IRMPD spectrum, including the bands of amide I and amide II. Conformations E, F, and G all partially predict the IRMPD bands. For the DapAlaH+ system shown in Figure 4, the lowestenergy conformation, U, seems to overestimate the amide I band but underestimate the amide II band. Instead, the second lowest-energy conformation, V, appears to have a good match to the overall IRMPD spectrum, including amide I and amide II. Conformation X also has good prediction for the bands of amide I and amide II but overestimates the vibration of the Cterminal carboxyl group. The other conformations in this set all partially predict the IRMPD spectrum well. The higher degree of broadening in the IRMPD spectrum of DapAlaH+ suggests a higher degree of flexibility of the conformations for this peptide ion. Closely examining the conformations shown in Figure 2, one can note that the shapes among the conformations representing DapAlaH+ (U−Z) are more diverse than those representing AlaDapH+ (A−G). The overall analysis shows that each observed IRMPD spectrum is due to the vibrations of a population of low-energy conformations, and this contributes to the overall broadening of the spectrum. This effect is reflected in the Boltzmann-weighted IR spectra, which have an overall good match with the IRMPD spectra. 4.3. Characteristics of Structural Features and Molecular Properties. The intensity of the hydrogen bonding can be viewed in two ways, the strength of the interactions and the number of CO groups interacting with NH3+. Although the strength of the hydrogen-bonding interactions is not apparent by simply visualizing the conformations shown in Figure 2, the number of possible interactions can be compared. In general, the conformations of AlaDapH+ show a greater number of CO groups interacting with NH3+ than the conformations of DapAlaH+ do. This is especially visible when comparing the lowest-energy conformations (A vs U) for the peptide ions. The fact that these two conformations may not be the best representation of the peptide ions implies that the number of hydrogen bonds is not a main contribution to the intensity of the hydrogen-bonding interaction. Then what is the major contribution? The IRMPD spectral analysis suggests that conformations C and D are a better representation for AlaDapH+ and V and X are a better one for DapAlaH+. All of the four structures have two possible hydrogen-bonding interactions with one stronger (a shorter H···O distance) and one weaker (a longer H···O distance). Considering the stronger hydrogen-bonding interaction (Table 1), although the H···O distances appear similar, the bond angles are significantly different. In C and D, the angle of N−H···O is about 174° and the angle of H···OC is around 146−153°. Although in V and X, the angles of N−H···O and H···OC are around 147 and 125°, respectively. Larger angles in C and D suggest that the four atoms in moiety N−H···OC are closer to a linear alignment, whereas smaller angles in V and X indicate that the four atoms are in a curved alignment. As illustrated in Scheme 3, a hydrogen-bonding interaction can be viewed as dipole−dipole interaction between the N−H and the

Scheme 3. Model of Hydrogen-Bonding Interaction Illustrated as Dipole Vector Alignment

OC bonds. A linear alignment of the two bonds (or the four atoms) enables a favorable dipole−dipole interaction such that cancellation of the opposing dipole vectors becomes more effective. In the peptide ions, the NH3+ moiety contains a positive charge and the charge density is distributed over the three hydrogen atoms. Each N−H bond can be viewed as an effective dipole. Therefore, the linear alignment of the N−H and OC bonds allows an efficient sequestering of the positive charge at the NH3 moiety. This effect is likely the main contribution to the intensity of the hydrogen-bonding interaction at the OC group, which is responsible for the downshifting of the amide I band in AlaDapH+. As shown in Table 1, the angles of N−H···O and H···OC for AlaDapH+ are systematically larger than those for DapAlaH+ (although the averaged angles for N−H···O appear similar), suggesting a better alignment of the N−H and OC bonds for efficient hydrogen-bonding interaction in the former. Although each structure contains multiple hydrogen bonds, it appears that a single strong N−H···OC interaction is crucial for stabilizing the charge. In examining the overall dipole moments and the accessible surface areas of these four selected conformations (Table 1), it can be clearly seen that both C and D of AlaDapH+ have smaller dipole moment and smaller accessible surface area than V and X of DapAlaH+. In fact, all conformations of AlaDapH+ consistently have smaller dipole moments and smaller accessible surface areas than those of DapAlaH+. One can conclude that a favorable hydrogen bonding with strong charge−dipole interaction is thus responsible for the reduction of overall dipole moment, resulting in the more compact conformation. As mentioned in Introduction, among the two sets of the acetylated dipeptides containing an ionizable residue (X), the set with the ionizable residue at the C-terminus (AlaX) has a higher proton affinity than their counterparts with the ionizable residue at the N-terminus (XAla). Specifically, AlaDap has a proton affinity higher than that of DapAla by 2.5 kcal mol−1.24 It is clear that the higher proton affinity of AlaDap and of other peptides with the sequence AlaX is likely due to a single strong hydrogen-bonding interaction that effectively stabilizes the charge upon protonation of the peptides.

5. CONCLUSIONS The experimental IRMPD spectra for the two isomeric dipeptide ions, AlaDapH+ and DapAlaH+, are characterized in detail by comparing them with the theoretical IR spectra of a I


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(2) Kobylarz, M. J.; Grigg, J. C.; Takayama, S.-i. J.; Rai, D. K.; Heinrichs, D. E.; Murphy, M. E. P. Synthesis of L-2,3-diaminopropionic acid, a siderophore and antibiotic precursor. Chem. Biol. 2014, 21, 379−388. (3) Sasaki, N. A.; Garcia-Alvarez, M. C.; Wang, Q.; Ermolenko, L.; Franck, G.; Nhiri, N.; Martin, M.-T.; Audic, N.; Potier, P. N-Terminal 2,3-diaminopropionic acid (Dap) peptides as efficient methylglyoxal scavengers to inhibit advanced glycation endproduct (AGE) formation. Bioorg. Med. Chem. 2009, 17, 2310−2320. (4) Audic, N.; Potier, G.; Sasaki, N. A. New 2,3-diaminopropionic acid inhibitors of AGE and ALE formation. Org. Biomol. Chem. 2013, 11, 773−780. (5) Lan, Y.; Langlet-Bertin, B.; Abbate, V.; Vermeer, L. S.; Kong, X.; Sullivan, K. E.; Leborgne, C.; Scherman, D.; Hider, R. C.; Drake, A. F.; Bansal, S. S.; Kichler, A.; Mason, A. J. Incorporation of 2,3diaminopropionic acid into linear cationic amphipathic peptides produces pH-sensitive vectors. ChemBioChem 2010, 11, 1266−1272. (6) Dill, K. A.; MacCallum, J. L. The protein-folding problem, 50 years on. Science 2012, 338, 1042−1046. (7) Bartlett, G. J.; Porter, C. T.; Borkakoti, N.; Thornton, J. M. Analysis of catalytic residues in enzyme active sites. J. Mol. Biol. 2002, 324, 105−121. (8) Harris, T. K.; Turner, G. J. Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life 2002, 53, 85−98. (9) Cheng, X.; Wu, Z.; Fenselau, C. Collision energy dependence of proton-bound dimer dissociation: entropy effects, proton affinities, and intramolecular hydrogen-bonding in protonated peptides. J. Am. Chem. Soc. 1993, 115, 4844−4848. (10) Wu, Z.; Fenselau, C. Proton affinities of polyglycines assessed by using the kinetic method. J. Am. Soc. Mass Spectrom. 1992, 3, 863−866. (11) Cassady, C. J.; Carr, S. R.; Zhang, K.; Chung-Phillips, A. Experimental and Ab initio studies on protonations of alanine and small peptides of alanine and glycine. J. Org. Chem. 1995, 60, 1704− 1712. (12) McKieman, J. W.; Beltrame, C. E. A.; Cassady, C. J. Gas-phase basicities of serine and dipeptides of serine and glycine. J. Am. Soc. Mass Spectrom. 1994, 5, 718−723. (13) Kaltashov, I. A.; Fabris, D.; Fenselau, C. C. Assessment of gas phase basicities of protonated peptides by the kinetic method. J. Phys. Chem. 1995, 99, 10046−10051. (14) Ewing, N. P.; Zhang, X.; Cassady, C. J. Determination of the gas-phase basicities of proline and its di- and tripeptides with glycine: The enhanced basicity of prolylproline. J. Mass Spectrom. 1996, 31, 1345−1350. (15) Harrison, A. G. The gas-phase basicities and proton affinities of amino acids and peptides. Mass Spectrom. Rev. 1997, 16, 201−217. (16) Hahn, I.-S.; Wesdemiotis, C. Protonation thermochemistry of βalanine: An evaluation of proton affinities and entropies determined by the extended kinetic method. Int. J. Mass Spectrom. 2003, 222, 465− 479. (17) Bouchoux, G.; Desaphy, S.; Bourcier, S.; Malosse, C.; Bimbong, R. N. B. Gas-phase protonation thermochemistry of arginine. J. Phys. Chem. B 2008, 112, 3410−3419. (18) Schroeder, O. E.; Andriole, E. J.; Carver, K. L.; Colyer, K. E.; Poutsma, J. C. Proton affinity of lysine homologues from the extended kinetic method. J. Phys. Chem. A 2004, 108, 326−332. (19) Sunderlin, L. S.; Ryzhov, V.; Keller, L. M. M.; Gaillard, E. R. Measuring gas-phase basicities of amino acids using an ion trap mass spectrometer. A physical chemistry laboratory experiment. J. Chem. Educ. 2005, 82, 1071−1073. (20) Bouchoux, G.; Salpin, J.-Y. Gas-phase basicity of glycine, alanine, proline, serine, lysine, histidine and some of their peptides by the thermokinetic method. Eur. J. Mass Spectrom. 2003, 9, 391−402. (21) Sterner, J. L.; Johnston, M. V.; Nicol, G. R.; Ridge, D. P. Apparent proton affinities of highly charged peptide ions. J. Am. Soc. Mass Spectrom. 1999, 10, 483−491.

set of low-energy conformations. Although each IRMPD spectrum can be matched well with the IR spectra of a couple of lowest-energy conformations, the averaged IR spectra resulting from Boltzmann-weighted contributions of the set of low-energy conformations provide the closest match to the IRMPD spectra. The positions of the amide I and amide II bands in the two IRMPD spectra are notably different. Both amide I and amide II bands of AlaDapH+ appear to be downshifted compared to those of DapAlaH+, and the frequency difference between the two bands is smaller for the former. These spectral features suggest that the structure of AlaDapH+ has more intense hydrogen-bonding interaction between the amide carbonyl group (CO) and the charged NH3+ moiety than the structure of DapAlaH+. Detailed geometry analysis indicates that the stronger hydrogen-bonding interaction in the former is likely due to a better alignment of the N−H and OC bonds. This strong hydrogen bonding stabilizes the peptide ion by effectively sequestering the charge at the amino group. As a result, the structure of AlaDapH+ has a smaller dipole moment and is more compact. Stabilization of AlaDapH+ is associated with the higher proton affinity of AlaDap compared to DapAla. The overall results explain our previous observation well such that acetylated dipeptides with an ionizable residue at the Cterminus display greater proton affinity, which is associated with a smaller dipole moment and a more compact conformation of the protonated peptides.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b10435. Detailed geometry information for conformations A−G and U−Z (Figure S1); Boltzmann-averaged IR spectra calculated at MP2/6-311++G(2d,p)//ωB97X-D/6311+G(d) level of theory (Figure S2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: jren@pacific.edu. Phone: 209-946-2393. Fax: 209-9462607. ORCID

Giel Berden: 0000-0003-1500-922X Jianhua Ren: 0000-0001-6013-7066 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (CHE-1301505). The authors gratefully acknowledge the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) for the support of the FELIX Laboratory and highly appreciate the assistance by Juehan Gao and the FELIX staff. They also thank Yunato Zhang (University of the Pacific) for performing part of the calculations.

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REFERENCES

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