Vibrational Predissociation Spectroscopy of Cold Protonated

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A: Spectroscopy, Molecular Structure, and Quantum Chemistry

Vibrational Predissociation Spectroscopy of Cold Protonated Tryptophan With Different Messenger Tags Steffen Spieler, Chinh H. Duong, Alexander Kaiser, Felix Dünsing, Katharina Geistlinger, Moritz Fischer, Nan Yang, S. Sunil Kumar, Mark A. Johnson, and Roland Wester J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b07532 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

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The Journal of Physical Chemistry

Vibrational Predissociation Spectroscopy of Cold Protonated Tryptophan with Different Messenger Tags

Steffen Spieler†, Chinh H. Duong#, Alexander Kaiser†, Felix Duensing†, Katharina Geistlinger†, Moritz Fischer†, Nan Yang#, S. Sunil Kumar‡, Mark A. Johnson#, Roland Wester†*

†

Institut für Ionenphysik und Angewandte Physik, Universität Innsbruck, Technikerstraße 25, 6020 Innsbruck, Austria #

Sterling Chemistry Laboratory, Yale University, 225 Prospect St., New Haven, Connecticut 06520, United States

‡

Department of Physics, Indian Institute of Science Education and Research, Tirupati, Rami Reddy Nagar, Karakambadi Road, Mangalam (P.O.) Tirupati -517507, Andhra Pradesh, India * Corresponding Author: [email protected] Abstract Vibrational spectra of protonated tryptophan and microsolvated TrpH+(H2O)m=1,2 were recorded by predissociation of H2 messenger tags using cryogenic ion traps. We explore the issue of messenger induced spectral changes by solvating TrpH+(H2)n with n=1-5 to obtain single photon vibrational spectra of TrpH+ and of its partly deuterated isotopomer in the spectral region of 800-4400 cm-1. Depending on the number of messenger molecules, the spectra of several conformational isomers associated with multiple H2 binding locations along with two natural conformations of TrpH+ were found using the two photon MS3IR2 conformational hole burning method. Most probable messenger positions were established by comparison with predictions from DFT calculations on various candidate structures. Mechanical anharmonicity effects associated with the charged amino group were modeled by Born-Oppenheimer ab initio molecular dynamics. The spectra of TrpH+(H2O)m=1,2 reveal broad features in the NH stretching region of the NH3+ group, indicating strong hydrogen bonding in acceptor-donor configuration with the benzene ring for the first water molecule, while the second water appears to attach to a less strongly perturbing site, yielding unique transitions associated with the free OH stretching fundamentals. We discuss the structural deformations induced by the water molecules and compare our results to recent experiments on similar hydrated cationic systems.

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Introduction Spectroscopic studies of biological molecules have become an important source of information for their structure and thereby also their function in biological systems. Using vibrational spectroscopy in combination with electronic structure calculations the structure of single amino acids, small peptides, and DNA bases in the gas phase have been investigated. Also proton transfer and hydrogen bond cleavage or formation have received a lot of attention due to its importance in hydrated environments. In recent years, vibrational predissociation spectroscopy of ionic systems using messenger tagging has come into focus 1-7, due to its wide applicability to macromolecular systems especially when generated by electrospray ionization 8-9. These experiments benefit from the fact that the ion-tag complexes tend to dissociate after single photon excitation of the complex below the dissociation threshold of the bare ion. Messenger induced shifts can also be used to manipulate and thereby systematically study hydrogen bonded systems 8. The action spectroscopy technique in radio frequency traps effectively enables absorption experiments with a low number density of ions in the gas phase. In addition to messenger tagging spectroscopy, a range of action schemes have been developed such as laser-induced reactions 10, photodetachment of anions 11, infrared (multi-)photodissociation IR(M)PD 12, and laser-induced inhibition of cluster growth 13. Among these schemes, messenger tagging is applicable to a wide class of molecular ions irrespective of specific molecular properties. In contrast to free jet experiments, where the molecules are co-expanded with the messenger, messengers in ion traps can interact with mass-selected ions for longer interaction times. This yields a high tagging efficiency even for weakly bound complexes. Along with the low temperatures in the ion trap, the ion charge density and the induced or permanent dipole and quadrupole moments of the messenger are the most important parameters for this efficiency 10. For multiply tagged ions the combination of mass spectra and vibrational spectra can give insight into the size and structure of solvation shells around the core ion. Messenger tagging spectroscopy is faced by a central systematic uncertainty arising from the inevitable “matrix shift” due to the interaction between the ion and the tag. This shift is minimized with weakly interacting tags, such as helium or molecular hydrogen. To investigate it, one compares different tags or studies spectral features as a function of the number of tags. By extrapolation, the latter approach yields an approximate transition frequency of the tag-free ion and a good estimate of its systematic accuracy. Here we employ tagging spectroscopy in cryogenic ion traps to study the fundamental vibrational transitions of protonated tryptophan and the influence of different types and different numbers of tags. The amino acid tryptophan (Trp) has been the subject of a number of previous spectroscopic studies due to its biological relevance 11-14. It is an essential building block of proteins that can also serve as a structural marker due to its UV active side-chain, its chirality, and its resonances in electronic Raman scattering 14-17. Spectroscopic investigations of neutral tryptophan span the range from electronic 18-24 and ACS Paragon Plus Environment

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infrared vibrational 25-26, to fourier transform microwave spectroscopy of rotational transitions 27. Conformer specific spectra of Trp+ cations and its microsolvated analogues have been measured using resonant two-photon ionization, UV hole-burning spectroscopy, and infrared ion dip spectroscopy 28-29. The vibrational transitions of protonated tryptophan TrpH+ have been investigated for the first time by Mino et al. 12 using IRMPD in a room temperature Paul trap. They found sharp lines for the OH and NH stretches. Broad lines for the NH3+ modes were interpreted in terms of many structural conformers at room temperature with the common motif of NH3+ interacting both with the aromatic side chain and with CO from the carboxylic acid functionality. Recently, this system has been investigated with an IR/IR/UV scheme by Pereverzev et al. 30. Working with cryogenically cooled protonated tryptophan, they were able to establish that only two structural conformers are in play. In this work we present results from predissociation tagging spectroscopy of cryogenically cooled TrpH+, and compare them with those obtained using the IR/IR/UV scheme 30. We discuss the binding of up to five H2 messengers to the ion and their influence on the spectra. Using two color IR-IR hole burning spectroscopy, we confirm that two structural conformers are present in two different cryogenic trapping environments as described further below. Anharmonicities of the vibrational transitions are investigated by comparing the experimental spectra with static DFT and Born-Oppenheimer direct dynamics simulations. In addition, the structure of hydrated ions with one and two water molecules is addressed and compared with theoretical calculations. In this study we combine data from a newly built apparatus in Innsbruck, which combines electrospray ionization and cryogenic ion trapping in a multipole wire trap 31-32, with results obtained at the Yale MS3IR2 spectroscopy apparatus 33.

Experimental methods The sample of protonated tryptophan (TrpH+) was produced using a 1 mMol/l concentration of L-Tryptophan (Sigma Aldrich, reagent grade, ≥ 98%) in a 1:1 water-methanol solution. 1% acetic acid was added to enhance protonation. We recorded messenger tagged IR spectra with two different cryogenic experiments at Yale and Innsbruck, which are described briefly below. A. Yale MS3IR2 spectroscopy apparatus Tag predissociation spectroscopy and bare molecule IR2PD experiments were performed using the Yale custom time-of-flight temperature controlled cryogenic ion vibrational spectrometer 33. The ions are extracted from solution by electrospray ionization and then injected into the source region of the spectrometer. The TrpH+ cation was generated by electrospray ionization in a controlled N2 atmosphere. For the TrpH+(H2O)n=1,2 study, H2O was added to the spray chamber (i.e., before the ~0.5 mm capillary interface to the high ACS Paragon Plus Environment

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vacuum region of the instrument). These ions are then guided through several differential pumping stages with RF only octopole and quadrupole ion guides until they enter a 3-D Paul trap (Jordan TOF, Inc) that is mounted to a 4 K helium cryostat where they are accumulated for 95 ms. The temperature of the cryostat was held at 17 K for attachment of H2 molecules 34 and at 50 K to trap and cool hydrated complexes, that were produced in the electrospray process. During the accumulation time, the ions are cooled by collisions with helium buffer gas containing 15% H2 that is pulsed into the trap so that it can be evacuated prior to ion ejection. The H2 tagged or hydrated protonated tryptophan clusters are then pulsed out of the Paul trap and accelerated into the triple focusing time-of-flight photodissociation mass spectrometer, where they are mass-selected by a pulsed deflector and intersected by a tunable OPO/OPA infrared laser (LaserVision) to photoevaporate the H2 tag or water molecule. This photo fragmentation yield as a function of photon energy provides the vibrational spectrum. Further details associated with the instrument, in addition to the tagged, bare molecule IR2PD and double resonance depletion experiments are discussed in Ref. 33. B. Innsbruck cryogenic 16-pole wire trap apparatus A schematic of the Innsbruck ion trap setup is shown in Figure 1. Ions are produced by means of a home-built electrospray interface and are guided into a radio-frequency ion funnel 35 in vacuum. Two home-built differentially pumped quadrupoles guide the ions into our new 16-pole wire trap, a design motif taken from an octupole wire trap operated in our group 32, 36. Construction and properties of this ion trap will be addressed in a dedicated future publication, so we only provide a brief description here. Loading and trapping is optimized by two lens stacks with three ring electrodes that are mounted to the entrance and exit of the trap, giving an effective longitudinal trap length of 24.5 mm. Radial confinement is realized by 16 copper wires of 100 µm diameter. The housing of the trap is made out of a solid block of oxygen free copper to improve thermal conductance over all five trap walls. The trap is mounted on a 1 W closed cycle pulse tube cooler (Sumitomo SRP082E2) and can be temperature controlled by heating cartridges between 3 and 292 K. A home-built piezo valve provides buffer gas pulses, which are collisionally thermalized within a labyrinth structure inside the trap housing. To perform spectroscopy perpendicular to the axis at which the ions are loaded into the trap, the trap housing has one window on each side with large optical access. The trap is surrounded by a black body shield with optical access. Ions extracted from the trap enter an orthogonal Wiley-McLaren acceleration region 37 of a home built reflectron 38 time-of-flight detector. TrpH+, its most abundant fragment TrpH+(-NH3) and the protonated dimer Trp2H+ were simultaneously pre-trapped in the quadrupole guide before loading them into the cryogenic 16-pole wire trap, which was kept 13.5 K. Pure H2 buffer gas pulse was introduced from the piezo valve with a pulse width of about 2 ms. The ions were stored for 700 ms before extraction into the Wiley-McLaren region of a reflectron type time of flight detector. During the whole trapping procedure, a 1 kHz, 150 µJ tunable infrared Ekspla OPO system (NT277) ACS Paragon Plus Environment

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irradiated the trapped ions. Mass spectra were recorded as a function of OPO frequency at a rate of 1 Hz. To improve statistics, 60 mass spectra were recorded and averaged for the same OPO frequency. For each OPO frequency, the ion hits within single mass peaks were analyzed by summing over all hits within a 80 ns time window. The resulting total ion yield as a function of OPO frequency in the range of 2800-3700 cm-1 was background subtracted and averaged. This procedure gives the infrared spectra for individual mass peaks. The spectroscopic signal in the Innsbruck setup is derived from the laser-induced depletion of the parent ion signal inside the trap, while in the Yale setup, the signal is derived from the fragment yield measured on a zero background outside the trap. Thus, the Innsbruck data is derived from the difference signal with and without laser and has intrinsically a lower signal to noise ratio than the background-free signal of the Yale setup. However, the parent depletion approach allows one to record action spectra of different clusters or masses simultaneously, which is particularly suitable for measuring solvation effects for many different cluster sizes in parallel.

Computational methods The electronic ground state of protonated bare and tagged tryptophan was calculated with Kohn-Sham density functional theory at the B3LYP/6-31g(d,p) level/basis 39-42. H2 tags are primarily bound by charge-induced dipole interaction. Nevertheless, we included the Grimme D3 empirical dispersion correction to also account for van der Waals interactions 4243 . Various locally stable conformers and H2 binding positions were found by local geometry optimizations followed by harmonic frequency calculations as implemented in the Gaussian 09 and 16 programs44-45. The optimized geometries for the two protonated L-tryptophan conformers A and B are shown in Figure 2, and agree well with those identified earlier in Ref. 30 . The harmonic frequencies were scaled to match the indole NH stretch (factor 0.954) and then convoluted with Lorentzian functions for comparison with the experimental data. Tests with a triple-zeta basis set 6-311g++(2d, 2p) did not yield quantitatively better results. We also calculated the classical IR absorption spectra of the most stable conformers found in the optimizations to account for mechanical anharmonicity effects. In short, classical equations of motion are propagated numerically with forces calculated at each time-step by density functional theory46. The dipole moment of the ion can be obtained directly from the charge density at each time-step and the Fourier transformation of the dipole autocorrelation function yields the classical infrared absorption spectrum. We used the same level of theory as for the harmonic spectra, except that we changed to the correlationconsistent double-zeta cc-pVDZ basis set47. The trajectories were integrated with the conventional Velocity-Verlet algorithm with a time-step of 0.4 fs and propagated for 4-5 ps. The vibrational modes were initially excited so that the quantum numbers of each harmonic mode were occupied according to a thermal Boltzmann distribution at a vibrational


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temperature of 298 K. We used the VENUS 03 code for the calculations and refer to this method as Direct Dynamics (DD) 48-49.

Results and Discussion A. Assignment of the vibrational spectrum The single photon vibration predissociation spectrum of messenger H2 tagged protonated tryptophan is shown in Figure 3(a), taken with the Yale action spectrometer for a single H2 tag, and in Figure 3(b), taken with the Innsbruck ion trap setup for two H2 tags, in the range of 2800 to 3750 cm-1. The spectral resolution is limited in both cases by the OPO laser linewidth, which amounts to about 4 cm-1. Overall, both spectra agree very well with each other with respect to the line positions and also with respect to the line widths and band strengths. This already shows only a very small influence of the H2 tag. The subtle differences are discussed below. For comparison to the tag-free IR-UV method, the spectrum of Ref. 30 is shown in Figure 3(c). The assignments of the individual vibrational bands is straightforward based on previous work 30. The carboxyl OH stretching fundamental appears at 3560 cm-1 and that of the indole NH stretch at 3507 cm-1, as labeled in the Figure. The region between 2950 and 3400 cm-1 is dominated by three broad transitions of the protonated amino group. The NH3+ moiety contributes three IR-active modes with two asymmetric and one symmetric stretches. Harmonic DFT calculations yield for each normal mode a single NH stretch that has by far the largest vibrational amplitude. We label them with NH(α)-(γ), as assigned in Figures 2 and 3. The NH(α) bond interacts with the five-membered indole ring, which weakens it and red shifts the transition by nearly 300 cm-1 with respect to the free NH(γ) stretch. The NH(β) group forms a hydrogen bond to the carboxyl CO and is red shifted by 230 cm-1. The free NH(γ) centered at 3350 cm-1 is close to the asymmetric stretch fundamental (3340 cm-1) of bare NH4+ 50, and is therefore only weakly perturbed by the carboxyl group and the indole ring. There is, however, a (~22 cm-1) splitting in the NH(γ) band in the Yale spectrum, which is not observable in the Innsbruck spectrum. This doublet has been observed previously 30 and has been traced to two different conformers as discussed in the next section. In the spectral region of the NH3+ bands, the CH stretches of tryptophan may possibly also contribute to the observed structure. To test this possibility, we recorded the predissociation spectrum of partially deuterated TrpD+, which is displayed in Figure 5(d) and further discussed below. The four protons on the acidic -NH3+ and –COOH functional groups as well as that on the indole nitrogen were selectively isotopically substituted by spraying the compound in D2O and adding d3-acetic acid. The resulting spectrum of the heavy isotoplogue revealed that the CH transitions in the NH3+ region are weak and can safely be neglected when analyzing the NH3+ bands.


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The IRMPD studies of TrpH+ at room temperature 12 agree qualitatively well with the present spectra, but they also do not resolve the splitting in the NH(γ) feature. We also find good agreement with the recent IR/UV double resonance study 30, except that we do not find a splitting of the NH(β) band seen in that work. When comparing the absolute band positions of singly tagged TrpH+(H2) to the IR/UV results of bare TrpH+, we find OH 3560/3555, indole NH 3507/3503, NH(γ) 3360/3357 and 3338/3339, NH(β) 3120/(3123 and 3094) and NH(α) 3050/3044 30. For all transitions, our predissociation spectrum agrees within at most 6 cm-1 with the messenger free IR/UV spectrum. The average shift of our spectra with respect to the free IR/UV spectrum amounts to about 2 cm-1, which is estimated to be the systematic accuracy of the present frequency calibration. A feature that has not been explained thus so far is the rather broad, weak absorption centered around 3200 cm-1 in Figure 3(a). This was also seen, although less strongly, by Pereverzev et al. 30. In the Innsbruck spectrum in Figure 3(b) it is not evident. This feature may possibly be a Fermi resonance of an overtone of the amino umbrella with an NH stretch fundamental in analogy to the NH2 groups in neutral DNA 51. B. Conformer-resolved hole burning spectra The splitting in the NH(γ) transition in the TrpH+ spectrum (Figure 3a) has been attributed to two different conformers that provide different environments for the NH(γ) band 30. In conformer A, this band is in slight interaction with the indole ring, which can be observed by the red shift of the band, while it is far away from the indole ring in conformer B (see Figure 2), resulting in a high energy NH transition. To investigate the isomer contributions to the tagged spectra, we applied the MS3IR2 conformational hole burning technique 52-53 to singly H2 tagged TrpH+. One IR laser was scanned to generate an IR-modulated ion beam of both conformers. The TrpH+(H2) ion was mass-gated and specific conformers were probed by placing a second IR laser at a fixed photon energy of either 3341 cm-1 (for conformer A, red trace in Figure 3) or 3362 cm-1 (for conformer B, blue trace in Figure 3). The resulting IR-dip spectra are shown in Figure 3(d) and (f). Their differences, in particular the disappearance of the splitting of the free NH(γ) band, confirm the existence of the two conformers A and B in the tagged cations. Closer inspection reveals that conformer B shows a slight blue shift for indole-NH and OH in comparison to conformer A, which was not observed by Pereverzev et al. 30, and a marked red-shift of the NH(α) and NH(β) modes of ~9 cm-1 and ~16 cm-1 respectively. Adding up the spectra in Figure 3(d) and (f) reproduces the single-photon spectrum in Figure 3(a). However, the spectrum with two H2 tags (Figure 3(b)) resembles more closely the spectrum of conformer A than that of conformer B. This is most visible in the absence of the second NH(γ) band in Figure 3(b) and in the absence of the broad shoulder on the low frequency side of the NH(α) band. The same result is found in comparison with the doubly H2 tagged spectrum obtained with the Yale spectrometer (see Figure 4). We conclude from the fact that conformer A dominates for two H2 tags that this conformer binds H2 more strongly or kinetically favors attachment of the second H2. ACS Paragon Plus Environment

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The relative shifts of the NH bands between conformer A and B are reproduced by scaled harmonic spectra from DFT calculations (Figure 3(e), 3(g)). With a scaling factor of 0.954, both the NH(γ,indole) and OH stretch transitions match the simulations. Although the highly perturbed NH(α,β) transitions are calculated to be strongly red shifted in the simulations, the large observed red shifts (310 cm-1 and 240 cm-1) cannot be explained by interaction with the H2 messenger tags, as discussed in the next section. Instead, this suggests that anharmonic effects shift these transitions and also broaden them, which will be addressed in section D. C. Influence of the tag To investigate the influence of the messenger tags on the vibrational spectrum and the possible presence of different messenger isomers, the predissociation spectra of multiply H2 tagged TrpH+(H2)n, n=1-5 were recorded with the Innsbruck action spectrometer. The spectra are shown in the range of 2900 to 3700 cm-1 in Figure 4(a). The measured growth of bare TrpH+, shown for n=0 in Figure 4(a), represents the sum over all depletion signals. These spectra are compared to spectra for two and four H2 tags measured with the Yale spectrometer in the region of 2800 to 4200 cm-1 (see Figure 4(c)), which show the same trends for increasing cluster size. The shifts that are induced by the messenger tag are extracted by fitting normal distribution functions to the spectra in Figure 4(a). The result is plotted in Figure 4(b). While NH(α,β) experience a significant blue shift of ~10 cm-1, ~20 cm-1 and ~25 cm-1 for the second, third and fourth messengers, all other vibrational bands are only very weakly influenced by the H2 tags. For example, the carboxyl OH shows a small red shift of 2 cm-1, 1 cm-1 and 0.5 cm-1 for going from n=2 to 4. This is clear evidence that the first H2 tags bind to the NH3+ group of TrpH+. The fifth H2 tag no longer shifts the NH(α,β) bands, which indicates that now all direct tag binding sites are occupied and this H2 tag is either attached to the molecular backbone or in the second solvation shell. The exact position of the first H2 messenger cannot be extracted from the experimental data. The calculated structures for the two energetically close lying conformers A (+2.1 kJ/mol) and B (0 kJ/mol) (see Figure 2) accommodate the messenger tag at the benzene position (solid lines), the five-membered ring of the indole, or carboxyl sites (dashed lines), respectively. The calculated binding energies of the messenger are: i) 409 and 406 cm-1 at benzene, ii) 350 and 372 cm-1 at the five-membered ring, and iii) 250 and 188 cm-1 at the two available functional positions for conformers A and B, respectively. The higher binding energy and the better agreement with the vibrational spectra indicate that the benzene position is the most likely tagging position in both conformers (see Figure 2). The indole ring and functional positions are thus the most probable tag positions for the next two H2 tags that are bound to the NH3+ group. Tagging with three or more messengers produces a new feature at 3522 cm-1, indicating occupation of a messenger position directly on the carboxyl OH group (see both Figure 4(a) ACS Paragon Plus Environment

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and (c)). This shifts the free OH stretch by 40 cm-1 to the red in a similar manner as seen by Masson et al. 4 in protonated glycine. Upon interaction with the positively charged amino group, the H2 stretch fundamental becomes dipole allowed with a low-intensity peak appearing at 4120 cm-1 (see Figure 4(c)). It is 40 cm-1 red shifted from its unperturbed stretch fundamental. By adding more hydrogen messengers, the peak broadens asymmetrically and blue-shifts by 5 cm-1 upon addition of four H2 molecules. This supports the notion that the fourth H2 molecule attaches at a less attractive position than the first three. D. Anharmonicities Anharmonicities are usually large for vibrational bands of functional groups that contain a delocalized charge, as is the case for the protonated amino group of tryptophan-H+. Although the harmonic DFT simulations are in good qualitative agreement with the experimental findings (as discussed above), they cannot explain the strong anharmonic red shifts and broadenings of the NH(α,β) bands in the NH3+ group. In order to investigate the mechanical anharmonicity on the Born-Oppenheimer potential energy surface, ab initio molecular direct dynamics simulations (DD) 46 were carried out for both conformers A and B without messenger tags. In these classical trajectories, each vibrational mode was excited according to a thermal sampling from a Boltzmann distribution at Tvib = 298 K, while the initial angular momentum was chosen to be zero. The IR-spectrum was obtained from the dipole autocorrelation function. The results are compared to predissociation spectra of TrpH+(H2)1 and d5-TrpD+(H2)1 in Figure 5. For better comparison, the static DFT calculations are also shown. Overall, the DD results capture the red shifts in the experimental spectra without application of a scaling factor. Figure 5(a) and (d) displays the experimental spectra obtained with the Yale spectrometer over the range 800 to 4200 cm-1 for TrpH+ and from 1300 to 4200 cm-1 for d5-TrpD+. The DFT results in Figure 5(b) and (e) are shown in blue for conformer A and red for conformer B. Below 2000 cm-1, the most prominent feature is the carboxyl CO stretch at 1780 cm-1 followed by a weak unresolved NH3+ bend at 1600 cm-1. The NH3+ umbrella modes between 1400 and 1450 cm-1 are conformer dependent 30. Further to the red, the carboxyl COH bend transition at 1150 cm-1 splits into two bands. This splitting cannot be explained by messenger effects or conformational differences. The lowest observed vibration appears at 885 cm-1 and likely corresponds to a collective mode involving an asymmetric stretch of the central carbon atom connecting the functional groups with the side chain. In comparison, the measured vibrational frequency of the indole NH mode is captured by the DD calculations (Figure 5(c)) with an accuracy of 10 cm-1 and the carboxyl CO stretch within 15 cm-1. The carboxyl OH band is predicted to be 26 cm-1 blue shifted relative to the experimentally obtained value. This shift, as well as the broadening and the overestimated intensity indicate the complications arising from mechanical anharmonicity in this OH vibration. ACS Paragon Plus Environment

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The effect of anharmonicity is even stronger in the case of the NH3+ moiety. Here, no sharp features can be identified but a broad unresolved excitation continuum ranging from 2800 to 3250 cm-1, and another broad feature is observed near the NH(γ) mode. The anharmonic red shift of the NH(α,β) transitions with respect to the harmonic DFT calculations qualitatively capture the experimental trends. However, quantitative agreement is not reached, which is attributed to the limited statistics of the computationally costly DD simulations. In order to experimentally address the role of mechanical anharmonicity, the ion was partially deuterated such that all NH and OH hydrogens were replaced by deuterium isotopes. The measured spectrum is shown in Figure 5(d). The vibrational resonance around 2615 cm-1 includes both deuterated indole ND and the free OD stretch. A broad unresolved transition between 2170 and 2520 cm-1 contains all ND3+ (α,β,γ) modes. Only the carboxyl CO stretch at 1775 cm-1 can be assigned unambiguously by the harmonic DFT spectrum. In the region below 1500 cm-1, spectral congestion does not allow for clear assignment of the observed transitions. Overall, although DD captures all transitions and reproduces the broad feature of the ND transitions associated with the charged amino group, it overestimates the red shift of the free OD stretch. E. One- and two-water hydration

When attaching water instead of H2 as a tag, much stronger anharmonic effects are expected due to the short range ion-dipole interaction and strong hydrogen bonding. The vibrational spectra of TrpH+(H2O)n with n=1, 2 in the range of 2800 to 3800 cm-1 are shown in Figure 6. DFT calculations predict a binding energy of ~5700 cm-1 for the first water. Therefore, at least two photons are needed for dissociation of the cold ion. We find that the carboxyl OH and the indole NH stretches remain unchanged upon hydration with the first water molecule compared to the H2-tagged spectra of the bare ion. The monohydrate spectrum shows a feature at 3705 cm-1 close to the free OH stretching of a water molecule in an acceptor-donor configuration, where one hydrogen of the first water interacts strongly with the delocalized electrons of the benzene ring 54. The free NH(γ) transition is absent, which indicates that the first water molecules attaches at this position, causing a substantial red shift and/or intensity change of the transition. A broad unresolved feature is observed in the region 3100 to 3300 cm-1, reminiscent of the NH(α, β) transitions but with much lower intensity. It is likely that, since two photons are necessary for dissociation, the lower energy region of the spectrum below 3100 cm-1 is suppressed by the photodissociation kinetics. Upon addition of the second water, two new bands appear at 3648 cm-1 and 3744 cm-1. They are very close to the symmetric and asymmetric free water modes with redshifts of only 9 cm-1 and 12 cm-1 respectively. While the position of the carboxyl OH and indole NH remains unchanged, the free OH stretch broadens and loses intensity. Again, the free NH(γ) transition absent. However, a broad band can be observed below 3300 cm-1 with much higher intensity ACS Paragon Plus Environment

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than for the singly hydrated amino acid. Two unresolved features can be found at 3107 cm-1 and 3207 cm-1 which might be associated to the NH(α,β) transitions. The maximum of absorption is at ~2960 cm-1. Assignments based on harmonic calculations are difficult for the congested transitions associated with the protonated amino core ion. Thus, our modeling of the predissociation spectra by DFT methods focuses on the vibrations of the water ligands. Several structures of the complex were optimized and their scaled and normalized IR spectra were correlated to the experimental spectra in the region of 3300 to 3800 cm-1. We have tested eight different isomers with one water and nine different isomers with two waters. The computed correlation function of their simulated spectra with the experimental spectra allowed us to identify two isomers with one water and two isomers with two waters that agree well with the measured spectra (see Figure 7). For single water hydration we found reasonable agreement for the water oxygen bonding to NH(α), schematically shown in Figure 6 and 7(a). For the best correlated isomer (plotted opaque in Figure 7(a)), one hydrogen of water interacts with the delocalized electron cloud of the benzene ring, as also seen in experiments by Duncan and coworkers 55. While the first water stretch, associated with the free OH stretch in acceptor-donor configuration, lies at 3714 cm-1, the second water stretch is distorted by the strong interaction with the benzene ring. It was not observed in the spectra either because it coincidentally overlaps with the carboxyl OH or its intensity in a two photon process is too weak. The calculations also found a second isomer (plotted transparent in Figure 7(a)), where both hydrogens point away from the benzene ring. We infer that the interaction with the benzene ring is not strong enough to clearly differentiate these two isomers using the measured spectrum. NH(β) is blue shifted with respect to the hydrogen tagged bare protonated tryptophan by 100 cm-1 while NH(γ) experiences a red shift of 20 cm-1. DFT theory predicts the shared proton motion between the charge moiety and the water (NH(α)) to be at 2732 cm-1. The two best theoretical matches for the structure of the second water attached are shown in Figure 7(b). Both structures contain a water molecule bound to NH(α) and to the benzene ring. The calculations predict the second water to bind either to the NH(β), or to the free NH(γ). The experimental asymmetric and symmetric stretch of the second water are at 3730 and 3631 cm-1, respectively. Again, the symmetric water stretch of the first water attached to the NH(α) cannot be identified in the spectrum. While the blue shifted NH(β) is nearly unaffected upon hydration with the second water, the shared proton between NH(γ) and the second water is centered around 3032 cm-1 as the first water-NH(α) red shifts by 114 cm1 . This suggests that the filling of the hydration shell of protonated tryptophan starts at the core ion for the first and second water molecule and both water molecules interact with the amino group. Interestingly, although the interaction is much stronger for water than for H2, both tag species can be found at the same positions at cryogenic conditions.


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Conclusion We have conducted vibrational predissociation spectroscopy on protonated tryptophan complexed with up to five H2 messenger molecules as well as the first two hydrates. Two different low-energy conformers were identified using a MS3IR2 hole burning technique, which confirmed the results of Pereverzev et al. 30, who used a IR/IR/UV detection scheme. While the perturbation by the messenger molecules to the carboxyl OH and indole NH stretch is less than 5 cm-1, the modes associated with the charged NH3+ moiety shift by up to 12 cm-1 per attached messenger until the third messenger. The fourth messenger attaches to the carboxylic OH producing a second 40 cm-1 red shifted OH feature and the fifth messenger takes up a weakly interacting position, possibly in the second solvation shell. The positions of the reported vibrational bands are in good agreement with static DFT calculations using the conventional B3LYP method and a double-zeta 6-31g(d,p) basis set including polarization functions. The modes associated with the amino group are two to three times broader than the carboxylic OH and indole NH transitions. To investigate anharmonic effects the vibrational spectra were modeled using direct dynamics, which reproduces the experimental spectra qualitatively well over a wide range from 800-4200 cm1 . IR2PD spectra of singly and doubly hydrated protonated tryptophan show highly congested bands, indicating proton delocalization in a hydrogen bonded network. Here, the ligand structure of the complexed water molecules was investigated, which allowed us to identify the hydration motifs by comparison to DFT calculations. More work, in particular on linear spectra of multiply hydrated protonated tryptophan, is needed to allow for a quantitative description of the proton hydration dynamics in hydrated amino acids.

Acknowledgements We thank Bill Hase (Texas Tech University) for many fruitful discussions and for providing us with the software package VENUS interfaced with NWChem. The computational results presented have been achieved using the HPC infrastructure LEO of the University of Innsbruck. The Innsbruck work has been supported by the European Research Council under ERC grant agreement No. 279898. M. A. J. thanks the National Science Foundation under grant CHE-1465100 for funding the research on the protonated tryptophan and the Air Force Office of Scientific Research under grant FA9550-17-1-0267 for funding the two-color IR-IR triple focusing cryogenic photofragmentation mass spectrometer. C. H. D. thanks the National Science Foundation Graduate Research Fellowship for funding under Grant No. DGE-1122492.

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Figure 1: The new Innsbruck cryogenic 16-pole wire trap setup featuring the home built electro spray interface, an ion funnel, two quadrupole guides followed by the cryogenic 16pole wire trap. Details of the trap are shown in the inset figure. The trap is followed by a home built Wiley-McLaren reflectron type time of flight mass spectrometer.


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Figure 2: Optimized geometries of conformers A and B of protonated tryptophan. Various possible binding positions for the H2 tag are shown for conformer B. Each NH stretch of the protonated amino group can be distinguished and they are denoted by NH(α) interacting with the indole-ring, NH(β) hydrogen-bonding with CO, and free NH(γ).


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Figure 3: (a) Predissociation spectrum of singly H2 tagged TrpH+ measured at Yale (b) Predissociation spectrum of doubly H2 tagged TrpH+ measured in Innsbruck. (c) IR-UV data reproduced with permission from Ref. 30. (d), (f) Double resonance depletion spectra of TrpH+(H2)1, probed at 3341 cm-1 (red arrow) and 3362 cm-1 (blue arrow), respectively. (e), (g) Theoretical spectra of the two conformers A and B of TrpH+ with H2 tags at various positions, with the best agreement shown as solid lines and other positions shown as dashed lines.


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Figure 4: (a) Vibrational predissociation spectra of TrpH+(H2)n with n=0-5 obtained with the Innsbruck setup. Vertical dashed lines are plotted to show the messenger induced shifts of the specific vibrational bands. (b) Messenger induced line shifts for the specific bands as function of cluster size TrpH+(H2)n n=1-5. (c) Vibrational predissociation spectra of TrpH+(H2)n (n=1,2,4) obtained with the Yale setup.


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Figure 5: Experimental predissociation spectrum of TrpH+(H2) (a) and d5-TrpD+(H2) (d). The static DFT results for both conformers A and B of both species are shown in (b) and (e). Direct dynamics simulations at room temperature averaged over both conformers are shown in (c) and (f).


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Figure 6: IR2PD spectra of TrpH+(H2O)1 (a) and TrpH+(H2O)2 (c). Scaled static DFT calculations for the most probable hydration structure are shown in (b) and (d).


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Figure 7: DFT optimized structures of (a) TrpH+(H2O)1 and (b) and TrpH+(H2O)2. The structure with the best spectral overlap with the experiment is opaque and the next closest agreement is shown transparently.


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Vibrational Predissociation Spectroscopy of Cold Protonated

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