Probing Solvation-Induced Structural Changes in Conformationally

Subscriber access provided by UNIV OF LOUISIANA

A: Spectroscopy, Molecular Structure, and Quantum Chemistry

Probing Solvation-Induced Structural Changes in Conformationally Flexible Peptides: IR Spectroscopy of GlyH#(HO) 3

+

2

Kaitlyn C. Fischer, Jonathan M. Voss, Jia Zhou, and Etienne Garand J. Phys. Chem. A, Just Accepted Manuscript • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 21, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Probing Solvation-Induced Structural Changes in Conformationally Flexible Peptides: IR Spectroscopy of Gly3H+⋅(H2O)

Kaitlyn C. Fischer, Jonathan M. Voss, Jia Zhou, and Etienne Garand*

Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave, Madison, WI 53706

*email: [email protected]

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract IR predissociation spectroscopy of the Gly3H+(H2O) complex formed inside of a cryogenic ion trap reveals how the flexible model peptide structurally responds to solvation by a single water molecule. The resulting one-laser spectrum is quite congested, and the spectral analyses were assisted by both H2O/D2O substitution and IR-IR double resonance spectroscopy, revealing the presence of two contributing isomers and extensive anharmonic features. Comparisons to structures found via a systematic computational search identified the geometries of these two isomers. The major isomer, with all trans amide bonds and protonation on the terminal amine, represents ~90% of the overall population. It noticeably differs from the unsolvated Gly3H+, which exists in two isomeric forms, one with a cis amide bond and the other with protonation on an amide C=O. These results indicate that interactions with just one water molecule can induce significant structural changes, i.e. cis-trans amide bond rotation and proton migration, even as the clustering occurs within an 80 K cryogenic ion trap. Calculations of the isomerization pathways further reveal that the binding energy of the water molecule provides sufficient internal energy to overcome the barriers for the observed structural changes, and the minor solvation isomer results from a small fraction of the ions being kinetically trapped along one of the pathways.


Page 2 of 35

Page 3 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


I. Introduction Peptides and related biopolymers have considerable structural flexibility, allowing them to adopt different conformations in different environments. One of the main driving forces behind such structural changes is hydrogen-bonding (H-bonding), both intramolecularly and with the solvent environment1-2. On a larger scale, all of these interactions together result in the formation of secondary and tertiary structures of proteins that directly dictate their final functions. While there is a great deal of research on protein folding1, 3-5, the exact role of solvent in this process, albeit known to be crucial, is still not completely understood6-8. Spectroscopic studies on these systems are often complicated by spectral congestion and the presence of multiple isomers, limiting the information that can be extracted from the data. Hence, smaller peptides are used as model systems for detailed studies of intra- and intermolecular interactions, allowing for precise structural determinations and theoretical benchmarking. Moreover, spectroscopy of mass-selected clusters can probe the stepwise solvation of these peptides and reveal exactly how, for example, H-bonding can influence peptide structure. IR spectroscopy is a powerful technique for structural determination9-11 of isolated and microsolvated biomolecules because vibrational modes can be extremely sensitive to the local chemical environment. The experimental vibrational frequencies not only reveal the overall structure but can also highlight subtle changes after a perturbation, such as addition of a solvent molecule. Such studies of peptides have been carried out using infrared multiple photon dissociation (IRMPD) spectroscopy9, 11-15, UV-IR double resonance spectroscopy10, 16-18, and infrared predissociation (IRPD) spectroscopy19-22. Here, we use a combination of IRPD with isomer-specific IR-IR double resonance technique19, 23 and H2O/D2O substitution24 to reveal the



exact structural changes induced by the addition of a single water molecule to the protonated triglycine (Gly3H+) peptide. Previously, our IRPD study of the unsolvated Gly3H+ ion25, generated via electrospray ionization (ESI), identified the presence of two contributing isomers whose structure is shown in Figure 1. Both isomers contain a structural feature that is atypical in solution phase. The NcA isomer is protonated at the amine terminal but contains a cis amide bond, whose higher energy with respect to a trans amide is compensated by the

Figure 1. Structures of Gly3H+ identified in a previous study25. Relative energies (in kJ/mol, unscaled ZPE corrected) calculated at cam-B3LYP/def2TZVPP and MP2/def2TZVPP (in parentheses) are listed.

formation of a strong C=O···H-NH2 H-bond. The OtA isomer contains all trans amide bonds but is protonated at the C=O1 group rather than at the more basic -NH2 terminus. This structure is also stabilized by a strong intramolecular H-bond, in which the two amide C=O groups essentially share the proton. It appears that the peptide adopts these atypical configurations in the process of stabilizing the excess proton and calculations at different levels show disagreement on which structure is more stable. These distinctive gas phase structural characteristics immediately beg the question: how many solvent molecules are needed to establish the prevalent solution phase peptide structure? The spectroscopic results presented here show that complexation by a single water molecule is sufficient to yield a global minimum peptide geometry with a protonated amine and all trans amide bonds. While the Gly3H+(H2O) complex is still a relatively simple model system, its IRPD spectrum already exhibits several challenges which are expected to be common in the studies of microsolvated flexible molecules.


Page 4 of 35

Page 5 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


These challenges arise from the large number of possible energetically accessible structures, the increasing density of IR active modes, and the extensive anharmonic behaviors in the vibrational spectrum. We show that a combination of IR-IR double resonance spectroscopy19, 23 and H2O/D2O isotope substitution24 greatly facilitates the spectral analysis by piecewise decomposition of the one-laser spectrum.

II. Experimental details The IRPD spectra presented here were obtained using our homebuilt dual cryogenic ion trap vibrational spectrometer described in detail previously26. Briefly, ESI of a ~1 mM solution of triglycine dissolved in methanol with trace amounts of formic acid generated the Gly3H+ ions. From the source, ion guides transferred the ions through a series of differentially pumped regions into a linear octupole ion trap (reaction trap), held at 80 K by a liquid nitrogen cryostat. The ions inside the trap were thermalized via collisions with helium buffer gas, introduced in a ~1 ms burst at the beginning of the trap sequence. Usage of either H2O or D2O vapor seeded within the buffer gas further led to the formation of either Gly3H+(H2O) or Gly3H+(D2O) clusters. We have shown previously24 that under such cold clustering conditions, H/D exchange between ion and water molecule is almost entirely quenched. Thus, the mass-selected Gly3H+(D2O) clusters are expected to be minimally contaminated by isotopomers containing a partially deuterated peptide and HDO. The solvated clusters were then gently transferred into a 3D quadrupole ion trap (tagging trap) held at 10 K by a closed-cycle helium cryocooler, where they were further cooled and D2-tagged via collisions with helium buffer gas seeded with 10% D2. Finally, the tagged adducts were ejected into a time-of-flight (TOF) mass spectrometer, where they were mass-gated and intersected by the output of a 10 Hz Nd:YAG pumped tunable OPO/OPA laser (probe laser).



Resonant absorption of a single photon was sufficient to induce the loss of the weakly bound D2 tag, and the resulting photofragment ions were separated from the parent adducts by a two-stage reflectron. The one-laser IRPD spectra were obtained by monitoring the photofragment intensity while scanning the photon wavelength. Isomer-specific IR-IR spectra23 were obtained by focusing the output of a second Nd:YAG pumped tunable OPO/OPA laser (pump laser) directly into the 10 K tagging trap, ~90 ms after the introduction of the buffer gas. To acquire an ion-dip spectrum, the probe laser was fixed at a resonant frequency, and decreases in the photofragment yield were monitored while scanning the pump laser wavelength, resulting in an IR-IR spectrum that only contains contributions from isomers that have the probed vibration.

III. Computational details Computational structural searches were carried out using the molecular mechanics basin hopping program developed by the Hopkins group at the University of Waterloo.27-31 For each step taken, selected parameters were randomly changed within specified limits to generate a new structure which was then optimized using the AMBER force field. For example, starting with an optimized protonated straight-chain Gly3H+ solvated on the amine, the dihedral angles of the triglycine as well as the rotational and translational coordinates of the water molecule were iteratively stepped 10,000 times by random values between ±5°, ±5°, and ±0.1 Å, respectively. Any duplicate structures were rejected, and the search was repeated for different protonation and solvation sites. We also used NwcD, shown in Figure 2, as the starting structure with the dihedral of the cis amide bond fixed for a more focused search of structures containing this bonding configuration. Among the unique structures found, those within the lowest 4000 cm-1 energy


Page 6 of 35

Page 7 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


range were then further optimized via DFT calculations. Finally, structures containing a shared proton moiety, such as that found in OtA, were notably high in energy in the initial force field screening. These structures were specifically selected to be included in subsequent calculations. The Gaussian 16 program32 was used for DFT and MP2 calculations. Initial BLYP/3-21G optimizations filtered out duplicate structures and provided starting geometries for optimization using the cam-B3LYP functional, which has shown good agreements with experimental spectra for similar species23, 25-26. The ~100 lowest energy structures were first optimized with the 6-311G basis set and the ~60 lowest energy structures were further optimized with the def2TZVP basis set, yielding 45 unique structures. Harmonic IR spectra were calculated at the camB3LYP/def2TZVP level, which also provided unscaled ZPE corrections to the calculated energies. The harmonic frequencies were scaled by 0.954 in the 2400-3800 cm-1 region, determined using the experimental position of the Gly3H+ carboxyl OH stretch, and by 0.968 in the Figure 2: The 10 lowest energy structures of + Gly3H (H2O) optimized at the camB3LYP/def2TZVP level. Relative energies, ZPE corrected, are given in kJ/mol. MP2/def2TZVP energies, with DFT ZPE, are in parentheses.

1000-2400 cm-1 region based on the C=O stretches. The calculated spectra are gaussian broadened with the gaussian areas



corresponding to the calculated intensities. For strong H-bonded features, broader gaussian widths are used to facilitate comparisons to experimental spectra. These calculated results are summarized in Table S1 and Figures S1-S5. The geometries and energies of the ten lowest energy structures are shown in Figure 2. We name the structures using a similar scheme as Voss, et al25, based on the protonation site, configuration of the amide bonds, and a letter denoting the cam-B3LYP/def2TZVP ZPE corrected energetic ordering. This naming scheme generally groups the structures into three types: amine protonated isomers with all trans amide bonds (Nwt), amine protonated isomers with one cis amide bond (Nwc), and amide carbonyl protonated isomers with all trans amide bonds (Owt). The subscript “w” denotes that the peptide is solvated with a single water molecule, differentiating these structures from the bare Gly3H+ isomers. For the ten lowest energy structures shown in Figure 2, further geometry optimizations were carried out at the MP2/def2TZVP level. The MP2 energies, with cam-B3LYP/def2TZVP ZPE correction, showed a different ordering than the cam-B3LYP energies, and provide additional guidance when comparing relative stabilities between different structures. Note that for NwtD, MP2 optimization yielded the NwtC structure. Finally, the Owt structures are generally higher in energy than the amine protonated structures, with the lowest energy OwtA isomer calculated to be 17.3 kJ/mol (30.5 kJ/mol at MP2) above NwtA.

IV. Results The one-laser IRPD spectrum of Gly3H+(H2O).D2 is shown in Figure 3A. In the 31003800 cm-1 region, there are at least nine relatively intense and well-resolved features on top of many smaller ones, with the 3100-3400 cm-1 region being particularly congested. In addition,


Page 8 of 35

Page 9 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


there is a very broad feature spanning the 2400-3100 cm-1 region, pointing to the presence of a very strong H-bond33-36. The three O-H and five N-H bonds in a single Gly3H+(H2O) structure are insufficient to account for all these observed features in the 2300-3800 cm-1 region. Therefore, the one-laser IRPD spectrum indicates the presence of multiple isomers and/or anharmonic features such as combination and overtone bands. In comparison, the 1000-1900 cm-1 region is simpler. Between 1600-1900 cm-1, the Gly3H+(H2O).D2 spectrum displays four

Figure 3: One-laser IRPD spectra of (A) Gly3H+(H2O).D2 and (B) Gly3H+(D2O ).D2. (C) Overlay of (A) and (B) with the overlapping regions shown in purple.

dominant features, which can be accounted for by the three GlyH+ C=O stretches (two amide I and one carboxyl) and one water bending mode. To distinguish the vibrations originating from the water molecule from those of the Gly3H+ ion, we obtained the one-laser IRPD spectrum of Gly3H+(D2O).D2, shown in Figure 3B. Features belonging to H2O (blue), D2O (red), and the peptide (purple) are clearly highlighted when the two isotopologue spectra are overlaid, as shown in Figure 3C. This overlay shows that 9 ACS Paragon Plus Environment


many of the broadened features in the congested 3100-3400 cm-1 region are due to O-H stretches of H2O. In addition, the weak peaks at 3550 cm-1 and 3724 cm-1 and the intense feature at 3708 cm-1 also correspond to H2O O-H stretches. This is obviously more than the two expected stretch vibrations of H2O, indicating strong anharmonicity and/or the presence of multiple isomers. In the lower frequency region, isotope substitution indicates the 1638 cm-1 feature is likely the H2O bending mode. We can similarly analyze the D2O related features. First, there are the isotopically shifted modes corresponding to those identified above. For example, the free O-D stretches are expected to have frequencies around 2730 cm-1,37 and indeed we observe a broadened red feature centered at 2750 cm-1. However, there are also D2O (red) features present at 3103 cm-1, 3121 cm-1, and 3443 cm-1, which are above the highest possible frequency of an O-D stretch. Similarly, enhanced red intensity at 1774 cm-1 is too high to be the D2O bending mode which is expected to appear below 1200 cm-1.37 There are two possible explanations for the origin of these features. First, peptide modes may couple differently with H2O versus D2O modes due to their different frequencies. For example, this explains why the free OD stretch, overlapping in frequency with the broad feature spanning the 2400-3100 cm-1 region, is considerably broadened compared to the free OH stretch. The other explanation is that these extra red features arise due to changes in the relative populations of different isomers when the peptide is solvated with D2O rather than H2O. This is the more likely explanation for the features at 1774 cm-1, 3103 cm-1, 3121 cm-1, and 3443 cm-1. A similar population shift effect was observed in solvated GlyH+ clusters,24 and such behaviors are akin to solution phase observations that D2O can alter protein secondary structures and properties.38-39


Page 10 of 35

Page 11 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The possibility of multiple isomers contributing to the one-laser IRPD spectrum prompted us to carry out IR-IR ion-dip spectroscopy to disentangle contributions from individual structures. Surprisingly, IR-IR spectra with the probe laser fixed at 3174 cm-1, 3225 cm-1, 3292 cm-1, 3342 cm-1, 3363 cm-1, and 3403 cm-1 all have the same appearance, as shown in Figure 4B-G. Moreover, these spectra are very similar to the one-laser spectrum, with the most notable difference being the absence of the very weak OH stretch peaks at 3550 cm-1 and 3724 cm-1. Hence, the probed Figure 4: (A) One-laser IRPD spectrum of Gly3H+(H2O).D2. (B-H) correspond to the IR-IR ion dip spectra with the probe laser fixed on the indicated frequencies. (B) 3174 cm-1 (C) 3225 cm-1 (D) 3292 cm-1 (E) 3342 cm-1 (F) 3363 cm-1 (G) 3403 cm-1 (H) 3546 cm-1.

features in the 3170-3410 cm-1 region all belong to a single dominant isomer, and all of these observed H2O-related

intensities are a result of anharmonicity distributing the intensity of a single O-H oscillator into several combination bands. Such Franck-Condon-like progression typically arises from anharmonic coupling of the H-bonded O-H stretch with lower frequency modes.40-43 These bands are also in the spectral region of the H2O bend overtone (∼3250 cm-1) and may have additional complications arising from Fermi resonances44. The IR-IR spectrum acquired with the probe laser fixed at 3546 cm-1 reveals the minor but distinctive presence of another Gly3H+(H2O) isomer. For clarity, the spectrum shown in



Figure 4H has remnants of the major isomer removed. The dominant isomer has a small contribution here because the tail of its intense carboxyl O-H stretch at 3578 cm-1 extends into the weak probed feature. We note that this minor isomer exhibits a ~50 cm-1 width feature at 3110 cm-1 and a peak at 3443 cm-1, which correspond well to the D2O-related red features in Figure 3C that occur above the free O-D stretch, confirming that these features arise from a change in relative populations upon D2O substitution.

V. Spectral and structure assignments The experimentally identified Gly3H+ and H2O/D2O spectral features help us to narrow down the possible structures of the two isomers. We know that both isomers have a free H2O OH stretch peak above 3700 cm-1, so we can disregard any calculated structures where the H2O is a double H-bond donor, namely NwtC. Next, we consider the position of the H-bonded H2O O-H stretch determined using the H2O/D2O substitution. In the major isomer, this vibration is distributed over a ~200 cm-1 range with the most intense peak at 3225 cm-1, while it is observed at 3550 cm-1 for the minor isomer. The low energy calculated structures that show the best agreement with the above considerations are compared to the IRPD spectra in Figures 5 and 6. Based on the comparisons to calculations as shown in Figure 5, we assign the dominant isomer to NwtA, which is the lowest energy structure at both the cam-B3LYP/def2TZVP and MP2/def2TZVP levels of theory. In this structure, the water molecule accepts an H-bond from the protonated amine group and donates an H-bond to the C=O2 group, closely resembling the water binding motif observed in Gly2H+(H2O)26. The NwtB structure differs from NwtA by a rotation of the carboxyl terminus, and its calculated IR spectrum is similar to NwtA except for a lack of the ~3400 cm-1 peptide feature, which is blueshifted to overlap with the ~3470 cm-1 peak.


Page 12 of 35

Page 13 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5: One-laser IRPD spectrum of Gly3H+(H2O).D2, with the H2O features highlighted in blue, is compared with cam-B3LYP/def2TZVP calculated harmonic spectra, with the H2O vibrations highlighted in blue. The intensities of the two spectral regions are normalized independently.

The presence of this isomer cannot be fully ruled out, but the barriers for C-N bond rotation connecting NwtB to NwtA are calculated to be quite small (~0.2 kJ/mol), such that this higher energy structure is unlikely to be kinetically trapped in our experiment. The presence of the NwtD and NwtE isomers can be ruled out based on their poor agreement in the C=O stretch region compared to the experimental spectrum, as well as the ~3450 cm-1 feature that is absent in the experiment. The assignment of the dominant structure to NwtA allows us to make the following spectral assignments. The 3708 cm-1 feature is assigned to the H2O free O-H stretch and the distribution of intensities in the 3160-3400 cm-1 region is assigned to the H2O H-bonded O-H stretch. The feature at 3578 cm-1 is assigned to the carboxyl free O-H stretch while the features at 3479 cm-1 and 3403 cm-1 are assigned to the amide N2-H and N3-H stretches, respectively. For



Page 14 of 35

the protonated amine group, the feature at 3360 cm-1 is assigned to the free N-H stretch, the 3146 cm-1 feature is assigned to the stretch of the N-H that is interacting with C=O1, and the broad feature spanning the 2400-3000 cm-1 region is assigned to the N-H that is H-bond donating to the water molecule. Note that the 3146 cm-1 N-H stretch has a calculated frequency of 3215 cm-1, indicating some inaccuracies in describing the N1-H···O1=C interaction. In the lower frequency range, the feature at 1792 cm-1 is assigned to the carboxyl C=O3 stretch while the peaks at 1695 cm-1 and 1735 cm-1 are assigned to the amide I modes. Isotopic substitution points to the 1638 cm-1 feature as the H2O bending mode, ~20 cm-1 higher than the calculated frequency and overlapping it with one of the -NH3 scissoring modes. The -NH3 umbrella mode, on the other hand, is lower in frequency in the experiment than in the calculation, appearing at 1469 cm-1. Finally, the two amide II modes at ~1540 cm-1 also appear to have a larger splitting than predicted by calculation. These minor discrepancies hint at inaccuracies in the DFT calculations in capturing the strong H-bond between the protonated amine and the water molecule. For the minor isomer, the Hbonded H2O O-H stretch appears at 3550 cm-1, pointing to three possible low energy structures, shown in Figure 6. All three structures contain a cis O1CN2H amide bond and exhibit an Figure 6: IR-IR ion dip spectrum of the Gly3H+(H2O).D2 minor isomer, acquired with the probe laser fixed at 3546 cm-1, with the H2O stretch vibrations highlighted in blue, compared to camB3LYP/def2TZVP calculated harmonic spectra, with the H2O stretch vibrations highlighted in blue.

intramolecular interaction between the protonated amine and the C=O2 group.


Page 15 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The NwcD isomer exhibits three NH stretch vibrations near 3400 cm-1, in disagreement with the experimental spectrum, and therefore can be eliminated. The remaining NwcA and NwcC structures have almost identical calculated spectra in the high frequency range except for the ~2800 cm-1 H-bond donating amine N-H stretch, where NwcA spectrum shows better agreement with the IR-IR spectrum. Further support for assignment to NwcA can be found in Figure S6, where its C=O stretches show good agreement with the 1774 cm-1 feature that gains intensity in the Gly3H+(D2O) spectrum. The NwcA isomer is also the lowest energy structure considered here, hence, we assign it as the minor isomer. This allows us to make the following assignments. The 3724 cm-1 feature is assigned to the H2O free O-H stretch and the 3550 cm-1 peak is assigned to the H2O H-bonded O-H stretch. Note that the latter vibration is calculated 43 cm-1 lower in frequency, possibly indicating that the water H-bond donation to C=O1 is not as strong as predicted by theory in this complex. The feature at 3581 cm-1 is assigned to the carboxyl O-H stretch and the peaks at 3443 cm-1 and 3483 cm-1 are assigned to the N2-H and N3-H amide stretches, respectively. The features at 3330 cm-1 and 3110 cm-1 are assigned to the protonated amine N-H groups which are donating a H-bond to the carboxyl C=O3 and amide C=O2, respectively. The broad feature centered around 2780 cm-1 is assigned to the protonated amine N-H that is donating a H-bond to the water molecule. In the lower frequency region of the overall spectra, the feature at 1774 cm-1, which gains intensity upon D2O substitution, is assigned to the carboxyl C=O stretch of NwcA. Using the experimental peak areas and calculated intensities, we estimate that the NwtA structure accounts for ~90% of the total population in Gly3H+(H2O) and ~70% of the total population in Gly3H+(D2O) (see Figure S7 for details). We note that while these assignments explain all the features in the Gly3H+(H2O) spectrum, they do not account for the broad feature at



3268 cm-1 and the weak peak at 3701 cm-1 in the Gly3H+(D2O) spectrum. Since these features overlap with intensities in the Gly3H+(H2O) spectrum, they appear purple in the overlay spectra, as shown in Figure 3C and S8. The position of the 3701 cm-1 peak is characteristic of a water free O-H stretch and such a feature can only be the result of H/D exchange between D2O and Gly3H+, forming a (Gly3H+-d1)HDO cluster. Its slightly redshifted position with respect to the H2O free O-H stretch is consistent with the free O-H of an HDO. Similarly, the broad 3268 cm-1 feature may be due to the H-bonded O-H stretch of an HDO.

VI. Discussion It is interesting to compare the peptide structures found here with those identified previously for the desolvated Gly3H+ ion25, shown in Figure 1. IR-IR spectroscopy indicated that the Gly3H+ ion exists in an approximately 35:65 NcA:OtA ratio in our experiment. The minor Gly3H+(H2O) isomer, representing about 10% of the population, has a very similar peptide backbone structure as NcA, with protonation at the amine and the presence of a cis amide bond. As a result, the IRPD spectra of the minor NwcA isomer is quite similar to that of NcA (see Figure S9). In contrast, the dominant isomer found for Gly3H+(H2O) involves protonation at the amine and contains only trans amide bonds, unlike either NcA or OtA structures. We acquired the IRPD spectrum of the bare Gly3H+ ion again under the same trapping conditions as the solvated species, i.e., we probed the Gly3H+ population leftover from solvation clustering, and the resulting spectrum is identical to that published in Voss, et al.25. Hence, the NwtA structure most likely derives from conformational changes occurring from the solvation of both NcA and OtA structures and does not represent the preferential solvation of one structure. This result is somewhat surprising because rotating an amide bond or changing the protonation site is likely to


Page 16 of 35

Page 17 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


involve relatively high energy barriers. For example, the barrier for amide bond cis-trans isomerization in solution is on the order of 60-80 kJ/mol45-49. To shed further light on the solvation induced structural changes, we performed a series of calculations at the camB3LYP/def2TZVP level to find possible pathways connecting NcA and OtA to NwtA. The calculated NcA + H2O → NwtA pathway is shown in Figure 7A. The H2O binding energy in NwcA, the first minimum along the pathway, provides a sizeable 70 kJ/mol of internal energy towards isomerization. In addition, the thermal energy of the Gly3H+ NcA isomer is estimated to be 4 kJ/mol at 80 K, although this energy is not included in Figure 7. The highest barrier in this pathway, TSc2, involves the amide bond rotation and is located at -1.8 kJ/mol relative to NcA + H2O. Hence, the H2O binding energy alone provides sufficient energy for cis-trans isomerization. Since the clustering events occur in a collisional environment at 80 K, the observation of dominant NwtA populations in our experiment indicates that this isomerization must proceed rapidly Figure 7: Calculated reaction pathways for the solvation of NcA (A) and OtA (B) to form NwtA. The relative energies are calculated at the cam-B3LYP/def2TZVP level and includes unscaled ZPE correction.

after the water attachment, before any substantial collisional cooling can



occur. Any Gly3H+(H2O) cluster that failed to overcome the cis-trans barrier before significant thermalization would therefore be trapped in the NwcA form by the large TSc2 barrier. The proton migration pathway for OtA + H2O → NwtA is shown in Figure 7B. The H2O binding energy in OwtA is only 44 kJ/mol. However, this is again sufficient to overcome the barrier in this pathway, which is calculated at -7 kJ/mol relative to OtA + H2O. The smaller TSO1 barrier is also consistent with the absence of the OwtA isomer in our IRPD spectra. Moreover, this pathway provides a possible explanation for the small, but noticeable, amount of H/D scrambling product observed in the Gly3H+(D2O) IRPD spectrum, especially in comparison to the minimal H/D exchange that was previously observed for GlyH+ + D2O24. Specifically, during the proton migration from the amide carbonyl to the terminal amine, an H3O+ moiety is formed in the Gly3H+(H2O) complex. The formation of this D2OH+ moiety in the Gly3H+(D2O) complex, i.e. how the D2O inserts into OtA and/or which proton finally migrates to the amine, may play a dictating role in the final branching ratio of the HDO isotopomer in the NwtA population. Finally, we consider the increased relative population of the minor NwcA isomer in the Gly3H+(D2O) spectrum. Upon D2O substitution, TSc2 is calculated to lower slightly to -3.3 kJ/mol relative to NcA + D2O. This is inconsistent with an increase in kinetically trapped structures. Note that the barrier height, e.g. NwcA TSc2, grows larger by 0.2 kJ/mol upon D2O substitution, but this small change is likely insufficient to account for the observed population change. We also considered the relative free energies (∆G) at 400 K, which is equivalent to the 70 kJ/mol of H2O binding energy being transformed into Gly3H+(H2O) internal energy. For the Gly3H+(H2O) complex, the ∆G(NwtA - NwcA) is calculated to be -7.5 kJ/mol favoring NwtA at the MP2 level of theory. If equilibrium is established at 400 K, a Boltzmann distribution would


Page 18 of 35

Page 19 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


yield 10% population in NwcA. The Gly3H+(D2O) cluster does have a slightly smaller ∆G(NwtA NwcA) of -7.3 kJ/mol, but this isotopic difference is not large enough to account for the observed increase of NwcA in Gly3H+(D2O). Of course, the above considerations hinge on the accuracy of the calculated energy differences, but given these values, there is nothing obvious from the energetics perspective that would explain the D2O related population change. Another possible explanation is that the clustering conditions differ slightly between experiments since these settings are adjusted daily to maximize the yields of the desired species. If the presence of NwcA is controlled by a close competition between isomerization and thermalization processes, then the amount of kinetically trapped isomers would be highly dependent on the buffer gas pressure inside the reaction trap. Careful study of this competition will require direct measurements of the buffer gas pressure in the ion trap as well as installation of an additional ion trap to prethermalize the ions before they are exposed to clustering conditions. Such improvements may also provide better control over the structures produced by the clustering process.

VII. Conclusions The Gly3H+(H2O) complex was spectroscopically probed to study how interactions with H-bonding solvent molecules can influence the structure of small flexible peptides. Although this is a relatively small complex, it exhibits spectral congestions that are likely to affect larger solvated complexes as well. Our results here showcase how IR-IR isomer-specific spectroscopy and H2O/D2O substitution can aide in the experimental analysis of the one-laser IRPD spectrum. Specifically, experimental results revealed the presence of two contributing isomers as well as signatures of anharmonic bands that contribute to significant spectral complexity. Comparisons with calculated structures showed that the dominant Gly3H+(H2O) isomer, NwtA, has both amide



bonds in the trans configuration and protonation on the terminal amine. It represents ~90% of the population and is structurally different than either the NcA or OtA isomer of the unsolvated Gly3H+ ion. Although the NcA + H2O → NwtA pathway involves a significant amide bond rotation barrier, the binding energy of the water molecule is sufficient for the majority of the ions to isomerize to the minimum energy NwtA cluster structure. The presence of the minor NwcA isomer results from kinetic trapping along this pathway, and it is structurally very similar to the cis-amide containing NcA structure identified for the bare ion25. The OtA + H2O → NwtA pathway involves smaller barriers and weaker, though still sufficient, water binding energies. Hence no solvation isomers exhibiting an amide C=O1 protonation site were observed experimentally. These two pathways also show how the presence of water molecules in a collisional environment can provide a pathway for conversion between NcA and OtA structures via NwtA. Finally, our results here suggest that the formation of larger solvated clusters in a cryogenic ion trap can be a suitable approach for accessing peptide structures that better represent those found in solution. Moreover, instrumental improvements to provide more precise control of the clustering conditions in the reaction trap may help further reduce the presence of kinetically trapped gas-phase structures.

Supporting Information Table S1 and Figures S1-S9 are included.

Acknowledgment We would like to thank Scott Hopkins and his group for providing us use of their basin hopping program. We would especially like to thank Mike LeCours for running the initial basin hopping


Page 20 of 35

Page 21 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


calculations and providing support in running further calculations and Josh Featherstone for providing support with the user interface. This work was supported by the US Department of Energy, Office of Science, Basic Energy Sciences, under award no. DE-SC0010326. The computational resources used in this work are supported by National Science Foundation grant CHE-0840494.



References (1) Dill, K. A.; MacCallum, J. L. The Protein-Folding Problem, 50 Years On. Science 2012, 338, 1042-1046. (2) Pauling, L.; Corey, R. B.; Branson, H. R. The Structure of Proteins - 2 Hydrogen-Bonded Helical Configurations of the Polypeptide Chain. Proc. Natl. Acad. Sci. U. S. A. 1951, 37, 205211. (3) Bartlett, A. I.; Radford, S. E. An Expanding Arsenal of Experimental Methods Yields an Explosion of Insights into Protein Folding Mechanisms. Nat. Struct. Mol. Biol. 2009, 16, 582588. (4) Hartl, F. U.; Hayer-Hartl, M. Converging Concepts of Protein Folding In Vitro and In Vivo. Nat. Struct. Mol. Biol. 2009, 16, 574-581. (5) Ignatova, Z.; Gierasch, L. M. Monitoring Protein Stability and Aggregation In Vivo by Real-Time Fluorescent Labeling. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 523-528. (6) Frauenfelder, H.; Fenimore, P. W.; Chen, G.; McMahon, B. H. Protein Folding is Slaved to Solvent Motions. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 15469-15472. (7) Lucent, D.; Vishal, V.; Pande, V. S. Protein Folding Under Confinement: A Role for Solvent. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 10430-10434. (8) Vaiana, S. M.; Manno, M.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U. The Role of Solvent in Protein Folding and in Aggregation. J. Biol. Phys. 2001, 27, 133-145. (9) Polfer, N. C.; Oomens, J. Vibrational Spectroscopy of Bare and Solvated Ionic Complexes of Biological Relevance. Mass Spectrom. Rev. 2009, 28, 468-494. (10) Rizzo, T. R.; Stearns, J. A.; Boyarkin, O. V. Spectroscopic Studies of Cold, Gas-Phase Biomolecular Ions. Int. Rev. Phys. Chem. 2009, 28, 481-515. (11) Scuderi, D.; Correia, C. F.; Balaj, O. P.; Ohanessian, G.; Lemaire, J.; Maitre, P. Structural Characterization by IRMPD Spectroscopy and DFT Calculations of Deprotonated Phosphorylated Amino Acids in the Gas Phase. ChemPhysChem 2009, 10, 1630-1641. (12) Oomens, J.; Polfer, N.; Moore, D. T.; van der Meer, L.; Marshall, A. G.; Eyler, J. R.; Meijer, G.; von Helden, G. Charge-State Resolved Mid-Infrared Spectroscopy of a Gas-Phase Protein. Phys. Chem. Chem. Phys. 2005, 7, 1345-1348.


Page 22 of 35

Page 23 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13) Oomens, J.; Steill, J. D. The Structure of Deprotonated Tri-Alanine and Its a3- Fragment Anion by IR Spectroscopy. J. Am. Soc. Mass Spectrom. 2010, 21, 698-706. (14) Polfer, N. C. Infrared Multiple Photon Dissociation Spectroscopy of Trapped Ions. Chem. Soc. Rev. 2011, 40, 2211-2221. (15) Wu, R. H.; McMahon, T. B. Protonation Sites and Conformations of Peptides of Glycine (Gly1-5H+) by IRMPD Spectroscopy. J. Phys. Chem. B 2009, 113, 8767-8775. (16) Buchanan, E. G.; James, W. H.; Choi, S. H.; Guo, L.; Gellman, S. H.; Muller, C. W.; Zwier, T. S. Single-Conformation Infrared Spectra of Model Peptides in the Amide I and Amide II Regions: Experiment-Based Determination of Local Mode Frequencies and Inter-mode Coupling. J. Chem. Phys. 2012, 137, 094301. (17) Dean, J. C.; Buchanan, E. G.; Zwier, T. S. Mixed 14/16 Helices in the Gas Phase: Conformation-Specific Spectroscopy of Z-(Gly)n, n=1, 3, 5. J. Am. Chem. Soc. 2012, 134, 17186-17201. (18) Nagornova, N. S.; Rizzo, T. R.; Boyarkin, O. V. Exploring the Mechanism of IR-UV Double-Resonance for Quantitative Spectroscopy of Protonated Polypeptides and Proteins. Angew. Chem., Int. Ed. 2013, 52, 6002-6005. (19) Leavitt, C. M.; Wolk, A. B.; Fournier, J. A.; Kamrath, M. Z.; Garand, E.; Van Stipdonk, M. J.; Johnson, M. A. Isomer-Specific IR–IR Double Resonance Spectroscopy of D2-Tagged Protonated Dipeptides Prepared in a Cryogenic Ion Trap. J. Phys. Chem. Lett. 2012, 3, 10991105. (20) Leavitt, C. M.; Wolk, A. B.; Kamrath, M. Z.; Garand, E.; Van Stipdonk, M. J.; Johnson, M. A. Characterizing the Intramolecular H-bond and Secondary Structure in Methylated GlyGlyH+ with H2 Predissociation Spectroscopy. J. Am. Soc. Mass Spectrom. 2011, 22, 19411952. (21) Marsh, B. M.; Duffy, E. M.; Soukup, M. T.; Zhou, J.; Garand, E. Intramolecular Hydrogen Bonding Motifs in Deprotonated Glycine Peptides by Cryogenic Ion Infrared Spectroscopy. J. Phys. Chem. A 2014, 118, 3906-3912. (22) Marsh, B. M.; Zhou, J.; Garand, E. Vibrational Spectroscopy of Isolated Copper(II) Complexes with Deprotonated Triglycine and Tetraglycine Peptides. RSC Adv. 2015, 5, 17901795. (23) Voss, J. M.; Kregel, S. J.; Fischer, K. C.; Garand, E. IR-IR Conformation Specific Spectroscopy of Na+(Glucose) Adducts. J. Am. Soc. Mass Spectrom. 2018, 29, 42-50.



(24) Voss, J. M.; Fischer, K. C.; Garand, E. Accessing the Vibrational Signatures of Amino Acid Ions Embedded in Water Clusters. J. Phys. Chem. Lett. 2018, 9, 2246-2250. (25) Voss, J. M.; Fischer, K. C.; Garand, E. Revealing the Structure of Isolated Peptides: IRIR Predissociation Spectroscopy of Protonated Triglycine Isomers. J. Mol. Spectrosc. 2018, 347, 28-34. (26) Marsh, B. M.; Voss, J. M.; Garand, E. A Dual Cryogenic Ion Trap Spectrometer for the Formation and Characterization of Solvated Ionic Clusters. J. Chem. Phys. 2015, 143, 204201. (27) Wales, D. J.; Doye, J. P. K. Global Optimization by Basin-Hopping and the Lowest Energy Structures of Lennard-Jones Clusters Containing up to 110 Atoms. J. Phys. Chem. A 1997, 101, 5111-5116. (28) Lecours, M. J.; Chow, W. C. T.; Hopkins, W. S. Density Functional Theory Study of RhnS0,± and Rhn+10,± (n = 1–9). J. Phys. Chem. A 2014, 118, 4278-4287. (29) Campbell, J. L.; Yang, A. M.-C.; Melo, L. R.; Hopkins, W. S. Studying Gas-Phase Interconversion of Tautomers Using Differential Mobility Spectrometry. J. Am. Soc. Mass Spectrom. 2016, 27, 1277-1284. (30) Liu, C.; Le Blanc, J. C. Y.; Shields, J.; Janiszewski, J. S.; Ieritano, C.; Ye, G. F.; Hawes, G. F.; Hopkins, W. S.; Campbell, J. L. Using Differential Mobility Spectrometry to Measure Ion Solvation: An Examination of the Roles of Solvents and Ionic Structures in Separating Quinoline-Based Drugs. Analyst 2015, 140, 6897-6903. (31) Campbell, J. L.; Zhu, M.; Hopkins, W. S. Ion-Molecule Clustering in Differential Mobility Spectrometry: Lessons Learned from Tetraalkylammonium Cations and their Isomers. J. Am. Soc. Mass Spectrom. 2014, 25, 1583-1591. (32) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H., et al. Gaussian 16, Revision B.01; Gaussian, Inc. Wallingford, CT, 2016. (33) Buckingham, A. D.; Del Bene, J. E.; McDowell, S. A. C. The Hydrogen Bond. Chem. Phys. Lett. 2008, 463, 1-10. (34) Del Bene, J. E.; Jordan, M. J. T. Vibrational Spectroscopy of the Hydrogen Bond: An Ab Initio Quantum-Chemical Perspective. Int. Rev. Phys. Chem. 1999, 18, 119-162. (35) Iogansen, A. V. Direct Proportionality of the Hydrogen Bonding Energy and the Intensification of the Stretching v(XH) Vibration in Infrared Spectra. Spectrochim. Acta A 1999, 55, 1585-1612. 24 ACS Paragon Plus Environment

Page 24 of 35

Page 25 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36) Leavitt, C. M.; DeBlase, A. F.; Johnson, C. J.; van Stipdonk, M.; McCoy, A. B.; Johnson, M. A. Hiding in Plain Sight: Unmasking the Diffuse Spectral Signatures of the Protonated NTerminus in Isolated Dipeptides Cooled in a Cryogenic Ion Trap. J. Phys. Chem. Lett. 2013, 4, 3450-3457. (37) Shimanouchi, T., Tables of Molecular Vibrational Frequencies Consolidated Volume I. National Bureau of Standards: 1972; pp. 1-160. (38) Venyaminov, S. Y.; Kalnin, N. N. Quantitative IR Spectrophotometry of Peptide Compounds in Water (H2O) Solutions. II. Amide Absorption Bands of Polypeptides and Fibrous Proteins in α-, β-, and Random Coil Conformations. Biopolymers 1990, 30, 1259-1271. (39) Kalnin, N. N.; Baikalov, I. A.; Venyaminov, S. Y. Quantitative IR Spectrophotometry of Peptide Compounds in Water (H2O) Solutions. III. Estimation of the Protein Secondary Structure. Biopolymers 1990, 30, 1273-1280. (40) Heine, N.; Kratz, E. G.; Bergmann, R.; Schofield, D. P.; Asmis, K. P.; Jordan, K. D.; McCoy, A. B. Vibrational Spectroscopy of the Water-Nitrate Complex in the O-H Stretching Region. J. Phys. Chem. A 2014, 118, 8188-8197. (41) Myshakin, E. M.; Jordan, K. D.; Sibert, E. L.; Johnson, M. A. Large Anharmonic Effects in the Infrared Spectra of the Symmetrical CH3NO2-·(H2O) and CH3CO2-·(H2O) complexes. J. Chem. Phys. 2003, 119, 10138-10145. (42) Robertson, W. H.; Price, E. A.; Weber, J. M.; Shin, J. W.; Weddle, G. H.; Johnson, M. A. Infrared Signatures of a Water Molecule Attached to Triatomic Domains of Molecular Anions: Evolution of the H-Bonding Configuration with Domain Length. J. Phys. Chem. A 2003, 107, 6527-6532. (43) Zabuga, A. V.; Kamrath, M. Z.; Rizzo, T. R. Franck-Condon-like Progressions in Infrared Spectra of Biological Molecules. J. Phys. Chem. A 2015, 119, 10494-10501. (44) Tabor, D. P.; Kusaka, R.; Walsh, P. S.; Sibert, E. L.; Zwier, T. S. Isomer-Specific Spectroscopy of Benzene-(H2O)n, n=6,7: Benzene's Role in Reshaping Water's ThreeDimensional Networks. J. Phys. Chem. Lett. 2015, 6, 1989-1995. (45) Chen, J.; Edwards, S. A.; Grater, F.; Baldauf, C. On the Cis to Trans Isomerization of Prolyl-Peptide Bonds under Tension. J. Phys. Chem. B 2012, 116, 9346-9351. (46) Fischer, S.; Dunbrack, R. L.; Karplus, M. Cis-Trans Imide Isomerization of the Proline Dipeptide. J. Am. Chem. Soc. 1994, 116, 11931-11937.



(47) Mookherjee, A.; Van Stipdonk, M. J.; Armentrout, P. B. Thermodynamics and Reaction Mechanisms of Decomposition of the Simplest Protonated Tripeptide, Triglycine: A Guided Ion Beam and Computational Study. J. Am. Soc. Mass Spectrom. 2017, 28, 739-757. (48) Wedemeyer, W. J.; Welker, E.; Scheraga, H. A. Proline cis-trans Isomerization and Protein Folding. Biochemistry 2002, 41, 14637-14644. (49) Valiaev, A.; Lim, D. W.; Oas, T. G.; Chilkoti, A.; Zauscher, S. Force-Induced Prolyl Cis−Trans Isomerization in Elastin-like Polypeptides. J. Am. Chem. Soc. 2007, 129, 6491-6497.


Page 26 of 35

Page 27 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphics



Figure 1 81x32mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 28 of 35

Page 29 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2 82x133mm (300 x 300 DPI)



Figure 3 177x114mm (300 x 300 DPI)


Page 30 of 35

Page 31 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 82x109mm (300 x 300 DPI)



Figure 5 177x107mm (300 x 300 DPI)


Page 32 of 35

Page 33 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6 82x82mm (300 x 300 DPI)



Figure 7 82x122mm (300 x 300 DPI)


Page 34 of 35

Page 35 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of content figure 80x43mm (300 x 300 DPI)


Probing Solvation-Induced Structural Changes in Conformationally

Recommend Documents