No abstract

Rotational spectroscopy in combination with molecular beams and laser ablation (laser-ablation molecular-beam Fourier transform microwave (LA-MB-FTMW) spectroscopy) has proved to be successful in characterizing the conformers of natural amino acids. The procedure usually followed to assign and identify the different conformers of an amino acid from the rotational spectrum is described through the study of the natural amino acid L-threonine. The solid sample of L-threonine was vaporized by laser pulses, diluted in Ne and supersonically expanded between the mirrors of a Fabry-Pérot resonator where it was spectroscopically probed by microwave radiation. The rotational and nuclear quadrupole coupling constants extracted from the analysis of the rotational spectrum are directly compared with those predicted by ab initio methods to achieve the conclusive identification of seven different conformers. A complex hydrogen bonding network arises as a consequence of the polar side chain of threonine.

We explored the conformational landscape of the proteinogenic amino acid serine [CH2OHOCH(NH2)OCOOH] in the gas phase. Solid serine was vaporized by laser ablation, expanded in a supersonic jet, and characterized by Fourier transform microwave spectroscopy. In the isolation conditions of the jet there have been discovered up to seven different neutral (non-zwitterionic) structures of serine, which are conclusively identified by the comparison between the experimental values of the rotational and quadrupole coupling constants with those predicted by ab initio calculations. These seven forms can serve as a basis to represent the shape of serine in the gas phase. From the postexpansion abundances we derived the conformational stability trend, which is controlled by the subtle network of intramolecular hydrogen bonds formed between the polar groups in the amino acid backbone and the hydroxy side chain. It is proposed that conformational cooling perturbs the equilibrium conformational distribution; thus, some of the lower-energy forms are ''missing'' in the supersonic expansion.amino acids ͉ conformations ͉ laser ablation ͉ microwave spectroscopy ͉ supersonic expansion A mino acids are distinguished by an outstanding conformational flexibility originating from multiple torsional degrees of freedom, which makes folding and functionality of proteins possible (1). Whereas covalent forces determine the molecular skeleton, conformational isomerism is controlled by weaker nonbonded interactions within the molecule, especially hydrogen bonding. Amino acids are also very sensitive to the chemical medium chosen for their study. In traditional studies in the condensed phase, the extensive intermolecular hydrogen bond interactions fix amino acids as doubly charged zwitterions (2, 3), wiping out the conformational variety of these molecules. The intrinsic structural preferences of amino acids can be revealed only when they are studied as free species in the gas phase, where neutral forms (present in polypeptide chains) are the most stable in detriment of ionic or zwitterionic forms. Levy and coworkers (4) were the first to measure highly resolved electronic spectra of amino acids in the gas phase by seeding tryptophan in a supersonic expansion. Since then, different experimental methods have been used to investigate the electronic spectrum of amino acids and a variety of biological molecules in the gas phase (5-7). The electronic spectrum is interpreted with the help of theoretical calculations to identify different conformations with a high degree of confidence. However, the use of electronic spectroscopy is limited to favorable cases that present aromatic chromophores. Only three of the 20 natural amino acids have aromatic side chains that meet this criterion: phenylalanine, tyrosine, and tryptophan (4,(8)(9)(10)(11)(12)(13)(14).Microwave spectroscopy, often considered the most definitive gas-phase structural probe, can distinguish unambiguously between different conformational structures and provide accurate structural inform...

The recent emergence of laser ablation molecular beam Fourier transform microwave (LA-MB-FTMW) spectroscopy [1,2] has made possible the gas-phase study of solid biomolecules with high melting points. In this approach, solids are vaporized by a high-energy laser pulse, supersonically expanded into a vacuum chamber, and characterized by their rotational spectrum. Of the biomolecules that have been studied by this technique, aliphatic amino acids have received special attention because of the lack of experimental information and their biological relevance. Amino acids are well known to exist as zwitterions (NH 3 + CH(R)COO À ) in the solid state and in aqueous solution, [3] but in the gas phase they are in the canonical neutral form NH 2 CH(R)COOH. This form represents the best approximation for understanding the inherent properties of these building blocks which are responsible for the specific shape of proteins.[4] The supersonic jet of a LA-MB-FTMW spectrometer provides a collisionless environment where amino acids can be considered isolated, thus allowing the evaluation of the role of intramolecular interactions through the investigation of conformational preferences in the gas phase. Seven different structures were characterized in a recent study on serine.[5]Herein we present our results on the study of cysteine, which is the only coded amino acid with a thiol group in the side chain.The majority of amino acids (proteogenic [6][7][8][9][10][11][12][13] and nonproteogenic [14][15][16] ) that have been studied so far are a-amino acids with nonpolar groups in their side chains. Their conformational behavior is mainly controlled by the stabilization effects associated with the formation of the three possible intramolecular hydrogen bonds. In configuration I (the notation follows that adopted for other aliphatic a-amino acids), [10] a bifurcated amine-to-carbonyl hydrogen bond (NÀ H···O = C) and a cis-COOH arrangement are established. Configuration II exhibits an intramolecular hydrogen bond between the hydrogen atom of the hydroxy group and the lone pair of electrons on the nitrogen atom of the amino group (N···H À O), which requires a trans-COOH configuration. In configuration III, the intramolecular hydrogen bond links the amino group and the oxygen atom of the hydroxy group (NÀH···OÀH), thereby recovering the cis-COOH arrangement. In all cases, the existence of a nonpolar side chain was found to have a negligible influence on the conformational preferences, with the most stable conformer displaying a type I intramolecular hydrogen bond.Cysteine (CH 2 SHCH(NH 2 )COOH) is a natural amino acid found in high abundance in keratin, the main protein in nails, skin, and hair.[17] A polar side chain in an a-amino acid will bring about a whole new set of interactions with the polar groups (-NH 2 , -COOH) in the amino acid backbone. These interactions, along with the great torsional flexibility that is characteristic of amino acids, may give rise to a considerable number of conformational minima relatively close in energy...

The combination of Fourier transform microwave spectroscopy in a pulsed supersonic jet with laser ablation has made beta-alanine amenable to a structural study in the gas phase. Two new conformers of beta-alanine have been identified together with the two previously observed by McGlone and Godfrey [J. Am. Chem. Soc. 1995, 117, 1043]. The comparison between the experimental rotational and 14N nuclear quadrupole coupling constants and those calculated ab initio provide a definitive test for molecular structures and confirm unambiguously the identification of all conformers. For the two most abundant conformers, an intramolecular hydrogen bond between the amino group and carbonyl oxygen (N-H...O=C) is established, and the COOH adopts a cis-COOH configuration. The next conformer in order of abundance presents an O-H...N intramolecular hydrogen bond with a trans configuration for the COOH group. The high sensitivity of the experiment has allowed us to detect for the first time a conformer uniquely stabilized by an n-pi* hyperconjugative interaction between the nucleophile N: of the amino group and the pi* orbital at the carbonyl group. Partial conformational relaxation has been observed in the supersonic expansion.

The multiconformational landscape of the proteogenic amino acid aspartic acid, the simplest amino acid with two carboxylic groups, has been explored for the first time in the gas phase. Solid aspartic acid (m.p. > 300 degrees C) was vaporized by laser ablation and expanded in a supersonic jet where rotational spectra of various conformers were obtained by Fourier transform microwave spectroscopy. Six different conformers have been conclusively identified by their distinct rotational and (14)N nuclear quadrupole coupling constants. The relative stability of the conformers rests on a delicate balance of the different intramolecular hydrogen bonds established between the two carboxylic groups and the amino group.

The supersonic molecular beam (absorption millimeter wave and microwave Fourier transform) spectra of the 1:1 complex between pyrimidine and four isotopomers of water (H2O, HDO, D2O, and H2 18O) have been assigned. The derived moments of inertia are consistent with a planar (or nearly planar) structure of the adduct in which one hydrogen of the water molecule is bound to the nitrogen of the aromatic ring, and the “free” water hydrogen is entgegen to the ring. Only high level ab initio calculations (6-31G**+MP2-BSSE) account for the coplanarity of the water with the ring molecule. The hydrogen bond stretching force constant and the dissociation energy have been derived to be k s = 13.8 Nm-1 and Δ E = 21.4 kJ/mol, respectively.

Acetylcholine causes endothelium-dependent dilation of normal arteries in most animal species. The effect of acetylcholine on normal human coronary arteries is controversial. Pathologic studies and epicardial echocardiography have shown that diffuse atherosclerosis is often present despite angiographic evidence of discrete coronary artery disease (CAD). Therefore, we postulated that acetylcholine would cause vasoconstriction of coronary arteries that are angiographically normal in patients with CAD. Coronary artery diameter, measured by automated quantification of digitized cineangiograms, was determined before and after the intracoronary infusion of 0.2 mM acetylcholine at 0.8-1.6 ml/min. The diameter of stenotic or irregular segments of six atherosclerotic coronary arteries decreased from 1.80±0.42 mm before acetylcholine to 1.26±0.46 mm after acetylcholine (p=0.0025). Acetylcholine had a significantly diYferent effect on the diameter of two groups of coronary arteries that are angiographically normal. Acetylcholine caused a 0.16±0.09-mm increase in the diameter of 14 normal coronary arteries in patients without CAD, whereas it caused a 0.26+0.12-mm decrease in the diameter of 14 normal coronary arteries in patients with CAD (p <0.01). Thus, the normal response to intracoronary acetylcholine is vasodilation, suggesting that endotheliumderived relaxing factor is released from normal human coronary endothelium. The vasoconstrictive effect of acetylcholine in the angiographically normal coronary arteries of patients with CAD suggests the presence of a diffuse abnormality of endothelial function. (Circulation 1989;79:287-291) T here is growing evidence that acetylcholine and a number of other substances induce vasodilation by stimulating the release of an endothelium-derived relaxing factor (EDRF) from endothelial cells, whereas nitroglycerin and other endothelium-independent vasodilators induce vasodilation through the direct stimulation ofvascular smooth muscle."2,3 Thus, acetylcholine or aggregating platelets cause relaxation of arteries with an intact endothelium but cause contraction of arteries denuded of endothelium.1"2'3 An impairment of endotheliumdependent vasodilation has been observed in rabbits4 and monkeys5'6 that were fed an atherogenic diet. atherosclerosis and restored endothelium-dependent relaxation to normal.6 Conflicting data exist, however, regarding the existence of endothelium-mediated vasodilation in human coronary arteries. Several in vitro studies conclude that acetylcholine and carbachol, both muscarinic agonists, constrict human coronary arteries,7-10 whereas other studies suggest that acetylcholine dilates normal human coronary arteries.1" Similarly, a discrepancy exists regarding the effect of acetylcholine on human coronary arteries in vivo. After the intracoronary infusion of acetylcholine, Horio et al12 observed at least a 25% decrease in the diameter of normal or almost normal coronary arteries in 27 of 70 arteries, whereas Ludmer et al13 reported an 11% increase in the diame...