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.

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).

Cryogenic Ion Spectroscopy of a Singly Protonated Peptide DYYVVR: Locating Phosphorylation Sites of a Kinase Domain.

Cryogenic ion spectroscopy (CIS) was applied to singly protonated DYYVVR, a tryptic peptide that contains the two active tyrosine residues (Y980 and Y981) of the Janus kinase 3 (JAK3) kinase domain, together with its point mutants (Y980F and Y981F) and phosphorylated peptides (pY980, pY981, and pY980pY981). The two tyrosine chromophores showed distinguishable UV absorption bands at around 35 200 and 35 450 cm-1, respectively. By comparing with the point mutants, the lower electronic band was assigned to the absorption of Y981, and the higher one was assigned to Y980. When phosphorylated, the UV absorption of the phosphorylated chromophore shifts to higher energy above 36 500 cm-1 but the unphosphorylated chromophore gives the absorption in the same region. Conformer-specific IR spectroscopy and density functional theory (DFT) calculations were used to tentatively assign the structure of DYYVVR. Two conformations were found, where Y981 is solvated by the protonated side chain of arginine R984, and the orientation of the carboxylic OH of D979 was different between the two. It is demonstrated that CIS can be used to distinguish the two tyrosine chromophores and to locate the phosphorylation site of a kinase domain.