The Gas Phase Protonation Sites of Six Naturally Occurring Nicotinoids.

The gas phase protonation sites of six naturally occurring nicotinoids, namely nicotine (NIC), nornicotine (NOR), anabasine (ANB), anatabine (ANT), cotinine (COT), and myosmine (MYO), consisting of a common Pyridine and differing non-Pyridine rings, have been determined for the first time at the physiological temperature from cryogenic ion trap infrared spectroscopy and electronic structure calculations. The protonation site on either of these two rings is related to the nicotinoid's biological activity. At room temperature, NIC is a mixture of Pyridine and Pyrrolidine (non-Pyridine) protomers, whereas NOR, ANB, ANT, and COT are pure Pyridine protomers and finally MYO is mostly a Pyroline (non-Pyridine) protomer. The nearly planar structure of MYO-H+, induced by the presence of a conjugated π system and confirmed from calculations and the UV absorption spectra, breaks from the trends observed for NIC, NOR, and ANB, since its structure is drastically different from the structures of the other nicotinoids.

Affinity of Nicotinoids to a Model Nicotinic Acetylcholine Receptor (nAChR) Binding Pocket in the Human Brain.

The binding affinity of nicotinoids to the binding residues of the α4ß2 variant of the nicotinic acetylcholine receptor (nAChR) was identified as a strong predictor of the nicotinoid's addictive character. Using ab initio calculations for model binding pockets of increasing size composed of 3, 6, and 14 amino acids (3AA, 6AA, and 14AA) that are derived from the crystal structure, the differences in binding affinity of 6 nicotinoids, namely, nicotine (NIC), nornicotine (NOR), anabasine (ANB), anatabine (ANT), myosmine (MYO), and cotinine (COT) were correlated to their previously reported doses required for increases in intracranial self-stimulation (ICSS) thresholds, a metric for their addictive function. By employing the many-body decomposition, the differences in the binding affinities of the various nicotinoids could be attributed mainly to the proton exchange energy between the pyridine and non-pyridine rings of the nicotinoids and the interactions between them and a handful of proximal amino acids, namely Trp156, Trpß57, Tyr100, and Tyr204. Interactions between the guest nicotinoid and the amino acids of the binding pocket were found to be mainly classical in nature, except for those between the nicotinoid and Trp156. The larger pockets were found to model binding structures more accurately and predicted the addictive character of all nicotinoids, while smaller models, which are more computationally feasible, would only predict the addictive character of nicotinoids that are similar to nicotine. The present study identifies the binding affinity of the guest nicotinoid to the host binding pocket as a strong descriptor of the nicotinoid's addiction potential, and as such it can be employed as a fast-screening technique for the potential addiction of nicotine analogs.

Can Ag+ Permeate through a Potassium Ion Channel? A Bottom-Up Approach by Infrared Spectroscopy of the Ag+ Complex with the Partial Peptide of a Selectivity Filter.

Silver and silver ions have a long history of antimicrobial activity and medical applications. Nevertheless, the activity of Ag+ against bacteria, how it enters a cell, has not yet been established. The K+ channel, a membrane protein, is a possible route. The addition of a channel inhibitor (4-aminopyridine) to modulate the Ag+ uptake could support this view. However, the inhibitor enhances the uptake of Ag+, the opposite result. We have applied cold ion trap infrared laser spectroscopy to complexes of Ag+ and Ac-Tyr-NHMe (a model for GYG) which is a portion of the selectivity filter in the K+ channel to consider the question of permeation. With support from quantum chemical calculations, we have determined the stable conformations of the complex. The conformations strongly suggest that Ag+ would not readily permeate the K+ channel. The mechanism of the unexpected enhancement by the inhibitor is discussed.

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.

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).

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.

Can the Partial Peptide SIVSF of the ß2-Adrenergic Receptor Recognize Chirality of the Epinephrine Neurotransmitter?

Chirality plays an essential role in biological molecular recognition, such as neurotransmission. Here, we applied electrospray-cold ion trap spectroscopy to complexes of a partial binding motif SIVSF of a ß2-adrenergic receptor pocket with L- and D-epinephrine AdH+. The ultraviolet spectrum of the SIVSF-AdH+ complex changed drastically when L-AdH+ was replaced by its enantiomer. The isomer-selected infrared spectra revealed that D-AdH+ was bound to SIVSF by its protonated amino-group or a single catechol OH and induced nonhelical secondary structures of SIVSF. This is in sharp contrast to the helical SIVSF complex with L-AdH+, which is close to the natural binding structure with two catechol OHs binding in the receptor. This shows that a short pentapeptide SIVSF can distinguish the chirality of the ligand AdH+ as well as the receptor. This stereoselectivity is suggested to arise from an additional interaction involving the hydroxyl group on the chiral carbon.

Ion-peptide interactions between alkali metal ions and a termini-protected dipeptide: modeling a portion of the selectivity filter in K+ channels.

Potassium channels have the unique ability to allow the selective passage of potassium ions at near diffusion-free rates while inhibiting the passage of more abundant sodium ions. Local interactions between chemical functional groups and the ions are responsible for both selectivity and transport. As an initial step in characterizing these interactions, the structures of Na+ and K+ complexed to the Ac-Tyr-NHMe peptide have been determined from infrared laser spectroscopy and supporting ab initio calculations. Ac-Tyr-NHMe, a termini-protected peptide sequence, replicates the GYG portion of one of the four peptide chains comprising the selectivity filter of a K+ channel. This peptide contains two carbonyl groups, among the eight C[double bond, length as m-dash]O groups forming the S1 binding site of the selectivity filter. Three conformations have been identified for both ions by laser IR-IR double resonance methods. Two conformations have the ion bound to the two C[double bond, length as m-dash]O groups. The third conformation has, in addition, a cation-π interaction with the aromatic ring of tyrosine, i.e. tridentate binding. The relative contributions of the three conformers are approximately the same for K+Ac-Tyr-NHMe, while the tridentate conformer is preferred for Na+Ac-Tyr-NHMe. These differences will be discussed in the context of ion mobility and selectivity.

Probing chirality recognition of protonated glutamic acid dimers by gas-phase vibrational spectroscopy and first-principles simulations.

The homochirality of the amino acid metabolism still puzzles biochemists. Vibrational spectroscopy of mass-selected gas-phase amino acids and their clusters can precisely reveal their conformation and might ultimately help to decode the interactions responsible for chirality recognition. Infrared photodissociation (IRPD) and conformer-selective IR-IR hole burning spectra of protonated glutamic acid dimers (LL-/LD-Glu2H+) recorded in the fingerprint and XH stretch ranges (1100-1900 and 2600-3600 cm-1) provide direct insight into their stereospecific interactions. Glu2H+ dimers are generated by electrospray ionization and stored in a cryogenic quadrupole ion trap held at 10 K. The assignment of the IRPD spectra is supported by vibrational analysis using many-body dispersion-corrected hybrid density-functional theory. Sampling of the conformational space is accomplished by basin hopping and replica-exchange molecular dynamics simulations. The most stable LD-Glu2H+ dimer (LD1) is predicted to be more stable than the most stable LL-Glu2H+ dimer (LL1) by ΔE0 = 4.0 kJ mol-1, which relies on stronger secondary interactions in LD1 as demonstrated by the noncovalent interaction method. IR-IR hole burning spectroscopy reveals the coexistence of at least four LD-Glu2H+ and three LL-Glu2H+ conformers. Their IR-dip spectra are assigned to the most stable conformers at room and cryogenic temperature, revealing incomplete thermalization of the ions by kinetic trapping in the cold trap. We observe different population ratios of LL and LD conformers of Glu2H+, as revealed by specific νNH2 and νCO intensities (fingerprints of chirality recognition).

Molecular Recognition by a Short Partial Peptide of the Adrenergic Receptor: A Bottom-Up Approach.

Receptor-neurotransmitter molecular recognition is key for neurotransmission. Although crystal structures of the receptors are known, the mechanism for recognition is not clear. Reported here is the ultraviolet (UV) and infrared (IR) spectra of complexes between a partial peptide (SIVSF), mimicking the binding motif of a catechol ring in the adrenergic receptor, and various ligands. The UV spectra show that two isomers coexist in the complex of SIVSF with properly recognized ligands, such as protonated adrenaline (adrenalineH+ ). From IR spectra, they are assigned to catechol- and amino-bound structures. The catechol-bound structure is not observed when the ligand is replaced by nonproper molecules, such as noradrenalineH+ . The results suggest that SIVSF not only recognizes the catechol ring but can distinguish differences in the amine side chain. The method provides a new possibility for screening molecules as potential therapeutics for activating the receptor.

Anharmonic Vibrational Analyses of Pentapeptide Conformations Explored with Enhanced Sampling Simulations.

An accurate theoretical prediction of the vibrational spectrum of polypeptides remains to be a challenge due to (1) their conformational flexibility and (2) non-negligible anharmonic effects. The former makes the search for conformers that contribute to the spectrum difficult, and the latter requires an expensive, quantum mechanical calculation for both electrons and vibrations. Here, we propose a new theoretical approach, which implements an enhanced conformational sampling by the replica-exchange molecular dynamics method, a structural clustering to identify distinct conformations, and a vibrational structure calculation by the second-order vibrational quasi-degenerate perturbation theory (VQDPT2). A systematic mode-selection scheme is developed to reduce the cost of VQDPT2 and the generation of a potential energy surface by the electronic structure calculation. The proposed method is applied to a pentapeptide, SIVSF-NH2, for which the infrared spectrum has recently been measured in the gas phase with high resolution in the OH and NH stretching region. The theoretical spectrum of the lowest energy conformer is obtained with a mean absolute deviation of 11.2 cm-1 from the experimental spectrum. Furthermore, the NH stretching frequencies of the five lowest energy conformers are found to be consistent with the literature values measured for small peptides with a similar secondary structure. Therefore, the proposed method is a promising way to analyze the vibrational spectrum of polypeptides.

Gas phase ultraviolet and infrared spectroscopy on a partial peptide of ß2-adrenoceptor SIVSF-NH2 by a laser desorption supersonic jet technique.

Laser desorption supersonic jet laser spectroscopy has been applied to a penta-peptide, Ser-Ile-Val-Ser-Phe-NH2 (SIVSF-NH2), which is a partial sequence of a binding site in a ß2-adrenaline receptor protein. By comparing the resonance enhanced multiphoton ionization spectrum with the ultraviolet-ultraviolet hole burning (HB) spectrum, it is concluded that only a single conformer exists. The infrared (IR) spectrum of the X-H stretching region, measured by IR dip spectroscopy, shows that all of the OH and NH groups form hydrogen bonds. The structure of SIVSF-NH2 is assigned by the combination of a force field calculation (CONFLEX) and quantum chemical calculations both in S0 and S1. Over 20 000 stable conformations, given by CONFLEX, are classified into 6987 groups and 1068 groups in which all of the NH and OH bonds are hydrogen-bonded are selected. The most stable structure in each group was geometrically optimized by density functional theory (DFT) calculations, and theoretical IR spectra were calculated for the conformers for which the energies are within 10 kJ mol(-1) of the most stable one. It has been found that the most stable and the secondmost stable conformers well-reproduce the observed IR spectrum. The vibrational frequencies in S1 were also calculated for these two conformers. According to the reproduction of the vibrational frequencies in the HB spectrum, the structure of SIVSF-NH2 is assigned to the most stable conformer, which forms a hydrogen-bonded structure corresponding to a compact, distorted version of the beta hairpin of peptides and proteins.

Conformationally resolved spectra of acetaminophen by UV-UV hole burning and IR dip spectroscopy in the gas phase.

Electronic and vibrational spectra of acetaminophen were measured by using UV-UV hole burning (HB) and IR dip spectroscopy. HB spectra show the coexistence of 4 different species, which include two new ones. Low-frequency transitions in the spectra are reproduced by a one-dimensional periodic potential with a free-rotor basis set for the methyl group. From the analysis, we concluded that acetaminophen has two conformers and each conformer gives two independent transitions starting from the most stable 0a(1) and the hot 1e internal rotational levels. It is also found that the HB spectrum of the trans-conformer in the previous report is that from the 1e excited level, while the HB spectrum of the cis-conformer is contaminated by the transitions of the trans-conformer. Potential curves of the methyl rotational motion are determined both in S(0) and S(1).

Gas-phase spectroscopy of synephrine by laser desorption supersonic jet technique.

In our previous work, we found that synephrine has six conformers in the gas phase, while adrenaline, which is a catecholamine and has the same side chain as synephrine, has been reported to have only two conformers. To determine the conformational geometries of synephrine, we measured resonance enhanced multiphoton ionization, ultraviolet-ultraviolet hole burning, and infrared dip spectra by utilizing the laser desorption supersonic jet technique. By comparing the observed infrared spectra with theoretical ones, we assigned geometries except for the orientations of the phenolic OH group. Comparison between the determined structures of synephrine and those of 2-methylaminno-1-phenylethanol, which has the same side chain as synephrine but no phenol OH group, leads to the conclusion that the phenolic OH group in synephrine does not affect the conformational flexibility of the side chain. In the case of adrenaline, which is expected to have 12 conformers if there are no interactions between the catecholic OH groups and the side chain, some interactions possibly exist between them because only two conformations are observed. By estimation of the dipole-dipole interaction energy between partial dipole moments of the catecholic OH groups and the side chain, it was concluded that the dipole-dipole interaction stabilizes specific conformers which are actually observed.

Conformational reduction of DOPA in the gas phase studied by laser desorption supersonic jet laser spectroscopy.

The conformational reduction in catecholamine neurotransmitters was studied by resonance enhanced multi photon ionization (REMPI), ultraviolet-ultraviolet (UV-UV) hole burning and infrared (IR) dip spectroscopy with applying a laser desorption supersonic jet technique to DOPA, which is one of the catecholamine neurotransmitters and has one more phenolic OH group than tyrosine. It is concluded that DOPA has a single observable conformer in the gas phase at low temperature. Quantum chemical calculations at several levels with or without the dispersion correction were also carried out to study stable conformations. From the comparison between the computational IR spectra and the experimental ones, the most stable structure was determined. It is strongly suggested that the conformational reduction is caused by electrostatic interactions, such as a dipole-dipole interaction, between the chain and OH groups.

A new, highly sensitive time-of-flight mass spectrometer consisting of a flangeon-type conical ion lens system and a proto-type Daly detector for exhaust gas analysis based on the Jet-REMPI technique.

For the analysis of trace amounts of hazardous organic compounds, we developed a new ion detection system for supersonic jet resonance-enhanced multiphoton laser ionization mass spectrometry. High sensitivity and selectivity have been achieved by combining a proto-type Daly detector, a newly designed conical ion lens, and a potential switch that can perform the function of a mass selector. This ion detection system enables us to bring the jet nozzle closer to the ionization point. The detection sensitivity has thus been improved totally by more than 100 times compared with that obtained by the parallel-plate electrodes and micro-channel plate. We succeeded in assembling a flangeon-type ion extraction electrode consisting of a conical ion lens and a potential switch.