Switching of Protonation Sites in Hydrated Nicotine via a Grotthuss Mechanism.

The switching of the protonation sites in hydrated nicotine, probed by experimental infrared (IR) spectroscopy and theoretical ab initio calculations, is facilitated via a Grotthuss instead of a bimolecular proton transfer (vehicle) mechanism at the experimental temperature (T = 130 K) as unambiguously confirmed by experiments with deuterated water. In contrast, the bimolecular vehicle mechanism is preferred at higher temperatures (T = 300 K) as determined by theory. The Grotthuss mechanism for the concerted proton transfer results in the production of nicotine's bioactive and addictive pyrrolidine-protonated (Pyrro-H+) protomer with just 5 water molecules. Theoretical analysis suggests that the concerted proton transfer occurs via hydrogen-bonded bridges consisting of a 3 water molecule "core" that connects the pyridine protonated (Pyri-H+) with the pyrrolidine-protonated (Pyrro-H+) protomers. Additional water molecules attached as acceptors to the hydrogen-bonded "core" bridge result in lowering the reaction barrier of the concerted proton transfer down to less than 6 kcal/mol, which is consistent with the experimental observations.

Micro Solvation Effect of Methanol on Excited-State Dynamics of Protonated Tryptophan and Dopamine.

The electronic and vibrational cryogenic ion spectroscopy of protonated tryptophan (TrpH+) and dopamine (DAH+) complexed with methanol has been recorded. These two biological chromophores exhibit ultrafast photochemistry due to excited-state proton transfer (ESPT). We have established the relationship between the structure of the complexes and their photodynamics and compared them with recent results obtained in hydrated complexes. For TrpH+, there is no substantial change between methanol and water complexes; ESPT is hindered by a single solvent molecule. In the DAH+(MeOH)1 complex, the most stable conformer adopts a structure that prevents the direct interaction of the ammonium group of the side chain with the catechol ring, thus blocking the ESPT reaction. Such a ring structure is indeed a very minor populated conformer in the single-hydrated complex. The change in conformal stability between water and methanol clusters is due to a weak CH-π attractive interaction of the methyl group of methanol with the catechol.

Conformational preference of 2-(4-methoxyphenyl)ethanol studied by supersonic jet spectroscopy: Intramolecular OH/π interaction.

A π-type hydrogen bonding between the OH group and the π electron is a crucial factor for the conformational preference of the molecular structure with a flexible group. However, the information on the effect of the substituent on the OH/π interaction is insufficient. The laser-induced fluorescence (LIF) excitation, the dispersed fluorescence (DF), the IR-UV hole-burning, and the IR dip spectra of jet-cooled 2-(4-methoxyphenyl)ethanol were measured for the first time. Almost all bands observed in the spectral region of 35 550-36 500 cm-1 in the LIF excitation spectrum were successfully assigned with the DF and the IR-UV hole-burning spectra coupled with the quantum chemical calculation at M06-2x/6-311G and MP2/6-311G levels. Five conformers were found in the LIF excitation spectrum. The most stable conformer was Ggπ, and the second most stable conformer was Ggπ' (the trans rotamer of the methoxy group for Ggπ). Ggπ and Ggπ' had the OH group directed toward the π electron system of the benzene ring. The OH stretching frequency of Ggπ/Ggπ' of MPE in the IR dip spectra was red-shifted against that of Ggπ of phenylethanol, indicating that the introduction of the methoxy group would enhance the intramolecular OH/π interaction. In addition, the torsional vibration between the benzene ring and the side chain (-CH2CH2OH) (mode 63) was observed in the DF spectra of the Ggπ-00 and Ggπ'-00 band excitation, but their intensities were rather different, resulting from the different orientation of the OH group for each conformer toward the π electron system. The methoxy group would increase the negative charge on the benzene ring and would enhance the intramolecular OH/π interaction through the electrostatic interaction.

Does Chiral Sensitivity of a Structure Depend on the Metal Core? Alkali Ion Complexes of Cyclo(Tyr-Tyr).

Alkali metal complexes of cyclic dipeptide cyclo Tyr-Tyr have been studied under cryogenic ion trap conditions. Their structure was obtained by combining Infra-Red Photo-Dissociation (IRPD) and quantum chemical calculations. The structural motif strongly depends on the relative chirality of the tyrosine residues. For residues of identical chirality, the cation interacts with one amide oxygen and one of the aromatic rings only; the distance between the aromatic rings does not change with the nature of the metal. In contrast, for residues of opposite chirality, the metal cation is located in between the two aromatic rings and interacts with both of them. The distance between the two aromatic rings strongly depends on the metal. Electronic spectra obtained by Ultra Violet Photodissociation (UVPD) spectroscopy and analysis of the UV photo-fragments shed light on the excited state deactivation processes, which depend on both the chirality of the residue and that of the metal ion core. Na+ stands out by the presence of low-lying charge transfer states resulting in the broadening of the electronic spectrum.

Conformer-selective Photodynamics of TrpH+ -H2 O.

The photodynamics of protonated tryptophan and its mono hydrated complex TrpH+ -H2 O has been revisited. A combination of steady-state IR and UV cryogenic ion spectroscopies with picosecond pump-probe photodissociation experiments sheds new lights on the deactivation processes of TrpH+ and conformer-selected TrpH+ -H2 O complex, supported by quantum chemistry calculations at the DFT and coupled-cluster levels for the ground and excited states, respectively. TrpH+ excited at the band origin exhibits a transient of less than 100 ps, assigned to the lifetime of the excited state proton transfer (ESPT) structure. The two experimentally observed conformers of TrpH+ -H2 O have been assigned. A striking result arises from the conformer-selective photodynamics of TrpH+ -H2 O, in which a single water molecule inserted in between the ammonium and the indole ring hinders the barrierless ESPT reaction responsible for the ultra-fast deactivation process observed in the other conformer and in bare TrpH+ .

Hydration-induced protomer switching in p-aminobenzoic acid studied by cold double ion trap infrared spectroscopy.

Para-Aminobenzoic acid (PABA) is a benchmark molecule to study solvent-induced proton site switching. Protonation of the carboxy and amino groups of PABA generates O- and N-protomers of PABAH+, respectively. Ion mobility mass spectrometry (IMS) and infrared photodissociation (IRPD) studies have claimed that the O-protomer most stable in the gas phase is converted to the N-protomer most stable in solution upon hydration with six water molecules in the gas-phase cluster. However, the threshold size has remained ambiguous because the arrival time distributions in the IMS experiments exhibit multiple peaks. On the other hand, IRPD spectroscopy could not detect the N-protomer for smaller hydrated clusters because of broad background due to annealing required to reduce kinetic trapping. Herein, we report the threshold size for O → N protomer switching without ambiguity using IR spectroscopy in a double ion trap spectrometer from 1300 to 1800 cm-1. The pure O-protomer is prepared by electrospray, and size-specific hydrated clusters are formed in a reaction ion trap. The resulting clusters are transferred into a second cryogenic ion trap and the distribution of O- and N-protomers is determined by mid-IR spectroscopy without broadening. The threshold to promote O → N protomer switching is indeed five water molecules. It is smaller than the value reported previously, and as a result, its pentahydrated structure does not support the Grotthuss mechanism proposed previously. The extent of O → N proton transfer is evaluated by collision-assisted stripping IR spectroscopy, and the N-protomer population increases with the number of water molecules. This result is consistent with the dominant population of the N-protomer in aqueous solution.

Cation-responsive cavity expansion of valinomycin revealed by cryogenic ion trap infrared spectroscopy.

Valinomycin (VM) is a natural K+-selective ionophore that transports K+ through the cell membrane. VM captures K+ in its central cavity with a C3-symmetric ß-turn-like backbone. Although the binding affinity is drastically decreased for the VM-sodium (Na+VM) complex with respect to K+VM, VM holds relatively high affinity to Rb+ and Cs+. The high affinity for larger ions irrespective of ionic size seems to conflict with the expected optimal size matching model and raises questions on what factors determine ion selectivity. A combination of infrared spectroscopy with supporting computational calculations reveals that VM can accommodate larger Rb+ and Cs+ by flexibly changing its cavity size with the elongation of its folded ß-turn-like backbone. The high affinity to Rb+ and Cs+ can be ascribed to a size-dependent cavity expansion. These findings provide a new perspective on molecular recognition and selectivity beyond the conventional size matching model.

Stepwise hydration of [CH3COOMg]+ studied by cold ion trap infrared spectroscopy: insights into interactions in the magnesium channel selection filters.

The magnesium channel controls Mg2+ concentration in the cell and plays an indispensable role in biological functions. The crystal structure of the Magnesium Transport E channel suggested that Mg2+ hydrated by 6 water molecules is transported through a selection filter consisting of COO- groups on two Asp residues. This Mg2+ motion implies successive pairing with -OOC-R and dissociation mediated by water molecules. For another divalent ion, however, it is known that RCOO-⋯Ca2+ cannot be separated even with 12 water molecules. From this discrepancy, we probe the structure of Mg2+(CH3COO-)(H2O)4-17 clusters by measuring the infrared spectra and monitoring the vibrational frequencies of COO- with the help of quantum chemistry calculations. The hydration by (H2O)6 is not enough to induce ion separation, and partially-separated or separated pairs are formed from 10 water molecules at least. These results suggest that the ion separation between Mg2+ and carboxylate ions in the selection-filter of the MgtE channel not only results from water molecules in their first hydration shell, but also from additional factors including water molecules and protein groups in the second solvation shell of Mg2+.

Infrared Spectra of Beauvericin-Alkaline Earth Metal Ion Complexes─Ion Preference to Physiological Ions.

Beauvericin (Bv) is a naturally occurring ionophore that selectively transports ions through cell membranes. However, the intrinsic ion selectivity of Bv for alkaline earth metal ions (M2+) is yet to be established due to inconsistent results from condensed phase experiments. Based on fluorescence quenching rates, Ca2+ appears to be preferred while extraction experiments favor Mg2+. In this study, we apply cold ion trap─infrared spectroscopy to Bv-M2+ coupled with electrospray ionization mass spectrometry. The mass spectrum shows that Bv favors binding to physiologically active ions Mg2+ and Ca2+ although it can form complexes with all four alkaline earth metal ions. Infrared spectroscopy, as measured by the H2 tag technique, reveals that Bv binds Mg2+ and Ca2+ ions by six carbonyl oxygens in the center of its cavity. This observation is supported by theoretical calculations. Other alkaline earth metal ions are bound by three carbonyl groups at the amide face. This difference in configuration is consistent with the binding preferences for the alkaline earth metal ions.

Structure of Gas Phase Monohydrated Nicotine: Implications for Nicotine's Native Structure in the Acetylcholine Binding Protein.

We report a joint experimental-theoretical study of the never reported before structure and infrared spectra of gas phase monohydrated nicotine (NIC) and nornicotine (NOR) and use them to assign their protonation sites. NIC's biological activity is strongly affected by its protonation site, namely, the pyrrolidine (Pyrro-NICH+, anticipated active form) and pyridine (Pyri-NICH+) forms; however, these have yet to be directly experimentally determined in either the nicotinic acetylcholine receptor (nAChR, no water present) or the acetylcholine-binding protein (AChBP, a single water molecule is present) but can only be inferred to be Pyrro-NICH+ from the intermolecular distance to the neighboring residues (i.e., tryptophan). Our temperature-controlled double ion trap infrared spectroscopic experiments assisted by the collisional stripping method and high-level theoretical calculations yield the protonation ratio of Pyri:Pyrro = 8:2 at 240 K for the gas phase NICH+···(H2O) complex, which resembles the molecular cluster present in the AChBP. Therefore, a single water molecule in the gas phase enhances this ratio in NICH+ relative to the 3:2 for the nonhydrated gas phase NICH+ in a trend that contrasts with the almost exclusive presence of Pyrro-NICH+ in aqueous solution. In contrast, the Pyri-NORH+ protomer is exclusively observed, a fact that may correlate with its weaker biological activity.

A bottom-up approach to the ion recognition mechanism of K+ channels from laser spectroscopy of hydrated partial peptide-alkali metal ion complexes.

K+ channels allow selective permeation of K+, but not physiologically abundant Na+, at almost diffusion limit rates. The conduction mechanism of K+ channels is still controversial, with experimental and computation studies supporting two distinct conduction mechanisms: either with or without water inside the channel. Here, we employ a bottom-up approach on hydrated alkali metal complexes of a model peptide of K+ channels, Ac-Tyr-NHMe, to characterize metal-peptide, metal-water, and water-peptide interactions that govern the selectivity of K+ channels at a molecular level. Both the extension to the series of alkali metal ions and to temperature-dependent studies (approaching physiological values) have revealed the clear difference between permeable and non-permeable ions in the spectral features of the ion complexes. Furthermore, the impact of hydration is discussed in relation to the K+ channels by comparisons of the non-hydrated and hydrated complexes.

Collision-assisted stripping for determination of microsolvation-dependent protonation sites in hydrated clusters by cryogenic ion trap infrared spectroscopy: the case of benzocaineH+(H2O)n.

The protonation site of molecules can be varied by their surrounding environment. Gas-phase studies, including the popular techniques of infrared spectroscopy and ion mobility spectrometry, are a powerful tool for the determination of protonation sites in solvated clusters but often suffer from inherent limits for larger hydrated clusters. Here, we present collision-assisted stripping infrared (CAS-IR) spectroscopy as a new technique to overcome these problems and apply it in a proof-of-principle experiment to hydrated clusters of protonated benzocaine (H+BC), which shows protonation-site switching depending on the degree of hydration. The most stable protomer of H+BC in the gas phase (O-protonated) is interconverted into its most stable protomer in aqueous solution (N-protonated) upon hydration with three water molecules. CAS-IR spectroscopy enables us to unambiguously assign protonation sites and quantitatively determine the relative abundance of various protomers.

Gas phase protonated nicotine is a mixture of pyridine- and pyrrolidine-protonated conformers: implications for its native structure in the nicotinic acetylcholine receptor.

The infrared (IR) spectra of gas phase protonated nicotine has been measured in the never-before probed N-H "fingerprint region" (3200-3500 cm-1). The protonated molecules generated by an electrospray source are thermalized in the first ion trap with water vapor and He gas at a pre-determined temperature prior to being probed by IR spectroscopy in the second ion trap at 4 K. The IR spectra exhibit two N-H stretching bands which are assigned to the pyridine and pyrrolidine protomers with the aid of high-level electronic structure calculations. This finding is in sharp contrast to previous spectroscopic studies that suggested a single population of the pyridine protomer. The relative populations of the two protomers vary by changing the temperature of the thermalizing trap from 180-300 K. The relative conformer populations at 240 K and 300 K are well reproduced by the theoretical calculations, unequivocally determining that gas phase nicotine is a 3 : 2 mixture of both pyridine and pyrrolidine protomers at room temperature. The thermalizing anhydrous vapor does not result in any population change. It rather demonstrates the catalytic role of water in achieving equilibrium between the two protomers. The combination of IR spectroscopy and electronic structure calculations establish the small energy difference between the pyridine and pyrrolidine protomers in nicotine. One of the gas phase nicotine pyrrolidine protomers has the closest conformational resemblance among all low-lying energy isomers with the X-ray structure of nicotine in the nicotinic acetylcholine receptor (nAChR).

Excited state dynamics of protonated dopamine: hydration and conformation effects.

Electronic and vibrational spectroscopy in a cryogenic ion trap has been applied to protonated dopamine water clusters and assigned with the help of quantum chemistry calculations performed in the ground and electronic excited states. A dramatic hydration effect is observed when dopamine is solvated by three water molecules. The broad electronic spectra recorded for the bare and small water clusters containing protonated dopamine turn to sharp, well-resolved vibronic transitions in the 1-3 complex. This reflects the change induced by hydration in the photodynamics of protonated dopamine which is initially controlled by an excited state proton transfer (ESPT) reaction from the ammonium group toward the catechol ring. Interestingly, conformer selectivity is revealed in the 1-3 complex which shows two low lying energy conformers for which the ESPT reaction is prevented or not depending on the H-bond network formed between the dopamine and water molecules.

Do Stereochemical Effects Overcome a Charge-Induced Perturbation in Isolated Protonated Cyclo(Tyr-Tyr)?

Two diastereomers of the protonated diketopiperazine (DKP) dipeptide cyclo(Tyr-Tyr), namely, cyclo(LTyr-LTyr)H+ and cyclo(LTyr-DTyr)H+, are studied in a cryogenic ion trap by means of IR photodissociation spectroscopy combined with quantum chemical calculations. The two diastereomers have similar structures in which one of the rings is folded over the DKP ring and the other one is extended in a trans geometry, allowing a strong OH+···π interaction to take place. This contrasts to the observation of a stacked geometry for neutral cyclo(LTyr-LTyr) only under supersonic expansion conditions that do not exist for cyclo(LTyr-DTyr). In the protonated form, the strength of the OH+···π interaction is different for the two diastereomers, resulting in a ∼110 cm-1 difference in the ν(OH+) frequency and a smaller but clearly identifiable difference in the protonated amide ν(NH) frequency. Stereochemical effects are therefore still evidenced despite the strong perturbation due to the excess charge.

Potassium and sodium ion complexes with a partial peptide of the selectivity filter in K+ channels studied by cold ion trap infrared spectroscopy: the effect of hydration.

Potassium channels allow K+ to rapidly diffuse, while the selectivity filter (SF) actively blocks Na+. The presence of water in the SF during ion translocation remains under debate due to the experimental and computational challenges in characterizing the interactions between water, ions, and the SF. Our bottom-up approach has been applied to a system composed of a partial peptide of the SF (Ac-tyrosine-NHMe) with a metal ion and a single water molecule to probe these interactions. The IR photodissociation spectra of M+Ac-tyrosine-NHMe(H2O) (M = Na, K) combined with quantum chemical calculations revealed that the water molecule binding sites are ion-dependent. In addition, the ion-peptide distances are elongated significantly for the K+ complex in comparison to the Na+ complex by the addition of a single water molecule. This striking structural difference with the water molecule is discussed in relation to ion selectivity and translocation within the K+ channel.

Double Ion Trap Laser Spectroscopy of Alkali Metal Ion Complexes with a Partial Peptide of the Selectivity Filter in K+ Channels─Temperature Effect and Barrier for Conformational Conversions.

Potassium ion channels selectively permeate K+, as well as Rb+ and Cs+ to some degree, while excluding Na+ and Li+. Conformations of alkali metal complexes of Ac-Tyr-NHMe, a model peptide of the selectivity filter in a K+ channel, were previously found to correlate with the permeability of alkali metal ions to a K+ channel by cold ion trap infrared spectroscopy. With an additional temperature-controlled ion trap, we examined the conformations of the alkali metal complexes, allowing the ions to collide with a He buffer gas at different temperatures, prior to spectroscopic investigation. The conformational distribution of the K+-peptide complex shows the most significant variation with temperature, which suggests that this complex has more flexibility when complexed with K+ and suggests lower barrier heights than other metal-peptide complexes. The variability of the conformational distribution with temperature for the ions follows the same order of ion permeability of a K+ channel. This work demonstrates that the additional temperature-controlled ion trap is a powerful tool to explore the conformational landscape of flexible molecular systems.

Hydration-controlled excited-state relaxation in protonated dopamine studied by cryogenic ion spectroscopy.

Ultraviolet (UV) and infrared (IR) spectra of protonated dopamine (DAH+) and its hydrated clusters DAH+(H2O)1-3 are measured by cryogenic ion spectroscopy. DAH+ monomer and hydrated clusters with up to two water molecules show a broad UV spectrum, while it turns to a sharp, well-resolved one for DAH+-(H2O)3. Excited state calculations of DAH+(H2O)3 reproduce these spectral features. The conformer-selected IR spectrum of DAH+(H2O)3 is measured by IR dip spectroscopy, and its structure is assigned with the help of quantum chemical calculations. The excited state lifetime of DAH+ is much shorter than 20 ps, the cross correlation of the ps lasers, revealing a fast relaxation dynamics. The minimal energy path along the NH → π proton transfer coordinate exhibits a low energy barrier in the monomer, while this path is blocked by the high energy barrier in DAH+(H2O)3. It is concluded that the excited state proton transfer in DAH+ is inhibited by water-insertion.

Alkali and Alkaline Earth Metal Ions Complexes with a Partial Peptide of the Selectivity Filter in K+ Channels Studied by a Cold Ion Trap Infrared Spectroscopy.

The front cover artwork is provided by Takashi Tsujino (Science Graphics Co., Ltd.) . The image shows the efficacy of a bottom-up approach to ion selectivity of K+ channels. The GYG-K+ complex, which replicates the local portion of K+ channels, has three conformations with an equivalent distribution. Read the full text of the Article at 10.1002/cphc.202000033.

Alkali and Alkaline Earth Metal Ions Complexes with a Partial Peptide of the Selectivity Filter in K+ Channels Studied by a Cold Ion Trap Infrared Spectroscopy.

The infrared (IR) spectra of alkali and alkaline earth metal ion complexes with the Ac-Tyr-NHMe (GYG) peptide have been measured by laser photodissociation in a cold ion trap coupled with an electrospray mass spectrometer. The GYG peptide corresponds to a portion of the ion selectivity filter in the KcsA K+ channel that allows K+ to pass, but blocks Na+ even though it has a smaller ionic radius than K+ . This current study extends a previous investigation on Na+ and K+ to the entire set of alkali metaI ions and alkaline earth dications. IR-IR hole-burning (IR2 dip) spectroscopy has established the coexistence of several conformers of the GYG-metal ion complexes. The structures of the conformers were assigned by comparison between the isomer-selected IR spectra and theoretical IR spectra obtained from quantum chemical calculations. It was found that the structure of the dominant conformer correlates with the ability of the ion to permeate through the K+ channel.