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Revealing the Multiple Structures of Serine

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Revealing the Multiple Structures of Serine Revealing the multiple structures of serine Susana Blanco, M. Eugenia Sanz, Juan C. Lo´ pez, and Jose´ L. Alonso* Grupo de Espectroscopı´aMolecular, Departamento de Quı´micaFı´sicay Quı´micaInorga´nica, Universidad de Valladolid, Prado de la Magdalena s/n, E-47005 Valladolid, Spain Edited by Patrick Thaddeus, Harvard–Smithsonian Center for Astrophysics, Cambridge, MA, and approved October 4, 2007 (received for review June 20, 2007) We explored the conformational landscape of the proteinogenic solids with high melting points and low vapor pressures, and amino acid serine [CH2OHOCH(NH2)OCOOH] in the gas phase. consequently they are elusive to gas-phase investigations. There- Solid serine was vaporized by laser ablation, expanded in a fore, rotational studies of natural amino acids, started in the late supersonic jet, and characterized by Fourier transform microwave 1970s by Suenram and Godfrey (19–25) on glycine and alanine were spectroscopy. In the isolation conditions of the jet there have been restrained for a long time by the difficulties to bring these com- discovered up to seven different neutral (non-zwitterionic) struc- pounds into gas phase because they quickly decompose under the tures of serine, which are conclusively identified by the comparison classical heating methods of vaporization. We have recently revi- between the experimental values of the rotational and quadrupole talized this field with an experiment (see Fig. 1) that combines laser coupling constants with those predicted by ab initio calculations. ablation with Fourier transform microwave spectroscopy in super- These seven forms can serve as a basis to represent the shape of sonic jets (LA-MB-FTMW) (26). Amino acids are vaporized by the serine in the gas phase. From the postexpansion abundances we green line (532 nm) of a pulsed Nd:YAG laser (irradiances ϳ108 derived the conformational stability trend, which is controlled by W⅐cmϪ2), entrained in neon, and supersonically expanded into a the subtle network of intramolecular hydrogen bonds formed vacuum chamber, where they are probed by Fourier transform between the polar groups in the amino acid backbone and the microwave spectroscopy. The cooling afforded by the expansion, hydroxy side chain. It is proposed that conformational cooling which greatly simplifies the spectrum, and the extreme specificity to perturbs the equilibrium conformational distribution; thus, some molecular structure inherent to rotational spectroscopy make it of the lower-energy forms are ‘‘missing’’ in the supersonic possible to characterize the most abundant conformers in the expansion. supersonic expansion. Thus, their properties can be independently amino acids ͉ conformations ͉ laser ablation ͉ microwave spectroscopy ͉ studied, providing the finest structural information available in the supersonic expansion gas phase. In the last few years, this experimental approach has allowed us to explore the structural behavior of the ␣-amino acids alanine (27), valine (28), isoleucine (29), and leucine (30); the mino acids are distinguished by an outstanding conformational amino acids proline (31) and 4-hydroxyprolines (R- and S-) (32); the flexibility originating from multiple torsional degrees of free- A ␤-amino acid ␤-alanine (33); and other related ␣-amino acids dom, which makes folding and functionality of proteins possible (1). (34–36). These studies have shown that in the supersonic expansion BIOPHYSICS Whereas covalent forces determine the molecular skeleton, con- the simplest ␣-amino acids present two dominant conformers formational isomerism is controlled by weaker nonbonded inter- O ⅐⅐⅐ A actions within the molecule, especially hydrogen bonding. Amino stabilized by either a bifurcated N H O C hydrogen bond with a cis-COOH interaction (type I)oraN⅐⅐⅐HOO hydrogen bond (type acids are also very sensitive to the chemical medium chosen for their ⅐⅐⅐ study. In traditional studies in the condensed phase, the extensive II). Recently, we have even characterized the glycine H2O complex intermolecular hydrogen bond interactions fix amino acids as (37), which constitutes the first step of microsolvation of the doubly charged zwitterions (2, 3), wiping out the conformational simplest amino acid. The presence of polar functional groups in the side chains of variety of these molecules. The intrinsic structural preferences of CHEMISTRY ␣ amino acids can be revealed only when they are studied as free -amino acids is expected to dramatically increase the number of species in the gas phase, where neutral forms (present in polypep- low-energy conformers. This is the case of serine O O tide chains) are the most stable in detriment of ionic or zwitterionic [CH2OH CH(NH2) COOH], with a –CH2OH side chain. The forms. Levy and coworkers (4) were the first to measure highly hydroxyl group can establish additional intramolecular hydrogen resolved electronic spectra of amino acids in the gas phase by bonds as a proton donor to the amino group or to the carboxyl seeding tryptophan in a supersonic expansion. Since then, different group or as a proton acceptor through the nonbonding electron pair experimental methods have been used to investigate the electronic at its oxygen atom. These additional interactions, which do not spectrum of amino acids and a variety of biological molecules in the occur in other ␣-amino acids previously studied, may affect the gas phase (5–7). The electronic spectrum is interpreted with the conformational preferences and give rise to a rich conformational help of theoretical calculations to identify different conformations space. Indeed, a complete search of serine conformational space, with a high degree of confidence. However, the use of electronic carried out by Gronert et al. (38) by systematic rotations around the spectroscopy is limited to favorable cases that present aromatic five internal rotation axes, produced 51 conformational minima. In chromophores. Only three of the 20 natural amino acids have this work we have faced the challenge of investigating the multi- aromatic side chains that meet this criterion: phenylalanine, ty- conformational behavior of the neutral form of the ␣-amino acid rosine, and tryptophan (4, 8–14). serine using our LA-MB-FTMW technique. Microwave spectroscopy, often considered the most definitive gas-phase structural probe, can distinguish unambiguously between different conformational structures and provide accurate structural Author contributions: S.B., M.E.S., J.C.L., and J.L.A. designed research, performed research, information directly comparable to the in vacuo theoretical pre- analyzed data, and wrote the paper. dictions. Without any chromophore restriction, all amino acids are The authors declare no conflict of interest. potentially amenable to pure rotational studies. However, despite This article is a PNAS Direct Submission. significant instrumental advances in the microwave region carried *To whom correspondence should be addressed. E-mail: [email protected]. out in the last two decades (15–18), it is still difficult to extract This article contains supporting information online at www.pnas.org/cgi/content/full/ experimental information on the exact nature of amino acids’ 0705676104/DC1. conformations and their quantitative distributions. Amino acids are © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705676104 PNAS ͉ December 18, 2007 ͉ vol. 104 ͉ no. 51 ͉ 20183–20188 Downloaded by guest on September 30, 2021 to the hydrogen bonds established between the amino and carbox- ylic moieties, which we denoted as configuration I (NOH⅐⅐⅐OAC), configuration II (N⅐⅐⅐HOO), and configuration III (NOH⅐⅐⅐OOH), according to the convention used for smaller aliphatic amino acids. The different orientations of the side chain have been indicated by adding suffixes to the skeleton configuration i.e., a (corresponding to an ϩsc configuration for the –OH and –NH2 groups viewed along the C␤–C␣ bond, according to International Union of Pure and Applied Chemistry terminology), b (–sc), and c (ap). Additionally, a prime label indicates a down orientation of the –NH2 along with aOOH⅐⅐⅐N intramolecular interaction with the side chain. For configuration III additional ␣ or ␤ notation is used to indicate which H atom of the –NH2 binds to the –OH of the –COOH group (Fig. 2). Rotational Spectra. All of the serine structures of Fig. 2 are predicted to be near-prolate asymmetric tops, whose rotational spectra will show the characteristic patterns of the ␮a-type R-branch transitions. Initial scans were directed to search for the most intense rotational transitions of a particular conformer. 14N nuclear quadrupole hyperfine structure helped us to confirm that the observed lines belonged to one of the serine conformers. The presence of a single 14N nucleus (I ϭ 1) in serine splits each rotational transition into several close hyperfine components by the interaction of the 14N nuclear electric quadrupole moment with the molecular electric field gradient at the nitrogen position (40). A process of successive predictions and experimental measurements discarded or con- firmed the initial assignment until a final set of consistent rotational transitions was collected for each conformer. By using this proce- Fig. 1. Sketch of the LA-MB-FTMW spectrometer. dure, the detection of the most abundant conformers of serine could be readily accomplished. However, as the experimental work Results and Discussion proceeded to the observation of the least abundant species, very careful scans
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