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+.

Length-Dependent Transition from Extended to Folded Shapes in Short Oligomers of an Azetidine-Based α-Amino Acid: The Critical Role of NH···N H-Bonds.

Hydrogen bonds (H-bonds) are ubiquitous in peptides and proteins and are central to the stabilization of their structures. Inter-residue H-bonds between non-adjacent backbone amide NH and C=O motifs lead to the well-known secondary structures of helices, turns and sheets, but it is recognized that other H-bonding modes may be significant, including the weak intra-residue H-bond (called a C5 H-bond) that implicates the NH and C=O motifs of the same amino acid residue. Peptide model compounds that adopt stable C5 H-bonds are not readily available and the so-called 2.05-helix, formed by successive C5 H-bonds, is an elusive secondary structure. Using a combination of theoretical chemistry and spectroscopic studies in both the gas phase and solution phase, we have demonstrated that derivatives of 3-amino-1-methylazetidine-3-carboxylic acid, Aatc(Me) can form sidechain-backbone N-H···N C6γ H-bonds that accompany-and thereby stabilize-C5 H-bonds. In the capped trimer of Aatc(Me), extended C5/C6γ motifs are sufficiently robust to challenge classical 310-helix formation in solution and the fully-extended 2.05-helix conformer has been characterized in the gas phase. Concurrent H-bonding support for successive C5 motifs is a new axiom for stabilizing the extended backbone secondary structure in short peptides.

Characterization of Asx Turn Types and Their Connate Relationship with ß-Turns.

Models of asparagine-containing dipeptides specifically designed to favor intrinsic folding into an Asx turn were characterized both theoretically, by using quantum chemistry, and experimentally, by using laser spectroscopy in the gas phase. Both approaches provided evidence for the spontaneous folding of both the Asn-Ala and Asn-Gly dipeptide models into the most stable Asx turn, a conformation stabilized by a C10 H-bond that was very similar to a type II' ß-turn. In parallel, analysis of Asx turns implicating asparagine in crystallized protein structures in the Protein Data Bank revealed a sequence-dependent behavior. In Asn-Ala sequences, the Asx turn was found in conjunction with a type I ß-turn for which the first of the four defining residues was Asn. The observation that the Asx turn in these structures is mostly of type II' (i. e., its most stable innate structure) suggests that this motif might foster the formation and/or enhance the stability of the backbone ß-turn. In contrast, the Asx turns observed in Asn-Gly sequences extensively adopted a type II Asx-turn structure, thus suggesting that their formation should be ascribed to other factors, such as hydration. The fact that the Asx turn in a Asn-Gly sequence is also often found in combination with a hydrated ß-bulge supports the premise that a Asn-Gly sequence might efficiently promote the formation of the ß-bulge secondary structure.

Characterization of Asx Turn Types and Their Connate Relationship with ß-Turns.

Invited for the cover of this issue are David J. Aitken, Michel Mons, and co-workers at Université Paris-Saclay. The image depicts the investigation strategies used to document the intrinsic structures of an important secondary structure in proteins, the so-called Asx turn. Read the full text of the article at 10.1002/chem.202104328.

Neutral Peptides in the Gas Phase: Conformation and Aggregation Issues.

Combined IR and UV laser spectroscopic techniques in molecular beams merged with theoretical approaches have proven to be an ideal tool to elucidate intrinsic structural properties on a molecular level. It offers the possibility to analyze structural changes, in a controlled molecular environment, when successively adding aggregation partners. By this, it further makes these techniques a valuable starting point for a bottom-up approach in understanding the forces shaping larger molecular systems. This bottom-up approach was successfully applied to neutral amino acids starting around the 1990s. Ever since, experimental and theoretical methods developed further, and investigations could be extended to larger peptide systems. Against this background, the review gives an introduction to secondary structures and experimental methods as well as a summary on theoretical approaches. Vibrational frequencies being characteristic probes of molecular structure and interactions are especially addressed. Archetypal biologically relevant secondary structures investigated by molecular beam spectroscopy are described, and the influences of specific peptide residues on conformational preferences as well as the competition between secondary structures are discussed. Important influences like microsolvation or aggregation behavior are presented. Beyond the linear α-peptides, the main results of structural analysis on cyclic systems as well as on ß- and γ-peptides are summarized. Overall, this contribution addresses current aspects of molecular beam spectroscopy on peptides and related species and provides molecular level insights into manifold issues of chemical and biochemical relevance.

Stepwise dissociation of ion pairs by water molecules: cation-dependent separation mechanisms between carboxylate and alkali-earth metal ions.

Microhydrated H2-tagged ion pairs (Ca2+, AcO-)(H2O)n=0-8 and (Ba2+, AcO-)(H2O)n=0-5 are investigated by IR photodissociation laser spectroscopy and DFT-D frequency calculations. The detailed picture of the first steps of ion dissociation reveals two mechanisms, where water molecules promote dissociation either directly or indirectly depending on the nature of the cation.

Excited States Computation of Models of Phenylalanine Protein Chains: TD-DFT and Composite CC2/TD-DFT Protocols.

The present benchmark calculations testify to the validity of time-dependent density functional theory (TD-DFT) when exploring the low-lying excited states potential energy surfaces of models of phenylalanine protein chains. Among three functionals suitable for systems exhibiting charge-transfer excited states, LC-ωPBE, CAM-B3LYP, and ωB97X-D, which were tested on a reference peptide system, we selected the ωB97X-D functional, which gave the best results compared to the approximate coupled-cluster singles and doubles (CC2) method. A quantitative agreement for both the geometrical parameters and the vibrational frequencies was obtained for the lowest singlet excited state (a ππ* state) of the series of capped peptides. In contrast, only a qualitative agreement was met for the corresponding adiabatic zero-point vibrational energy (ZPVE)-corrected excitation energies. Two composite protocols combining CC2 and DFT/TD-DFT methods were then developed to improve these calculations. Both protocols substantially reduced the error compared to CC2 and experiment, and the best of both even led to results of CC2 quality at a lower cost, thus providing a reliable alternative to this method for very large systems.

Selenium in Proteins: Conformational Changes Induced by Se Substitution on Methionine, as Studied in Isolated Model Peptides by Optical Spectroscopy and Quantum Chemistry.

The side-chain of methionine residues is long enough to establish NH⋯S H-bonds with neighboring carbonyl groups of the backbone, giving rise to so-called intra-residue 6δ and inter-residue 7δ H-bonds. The aim of the present article is to document how the substitution of sulfur with a selenium atom affects the H-bonding of the Met system. This was investigated both experimentally and theoretically by conformation-resolved optical spectroscopy, following an isolated molecule approach. The present work emphasizes the similarities of the Met and Sem residues in terms of conformational structures, energetics, NH⋯Se/S H-bond strength and NH stretch spectral shifts, but also reveals subtle behavior differences between them. It provides evidence for the sensitivity of the H-bonding network with the folding type of the Sem/Met side-chains, where a simple flip of the terminal part of the side-chain can induce an extra 50 cm-1 spectral shift of the NH stretch engaged in a 7δ NH⋯S/Se bond.

Ion Pair Supramolecular Structure Identified by ATR-FTIR Spectroscopy and Simulations in Explicit Solvent*.

The present work uses ATR-FTIR spectroscopy assisted by simulations in explicit solvent and frequency calculations to investigate the supramolecular structure of carboxylate alkali-metal ion pairs in aqueous solutions. ATR-FTIR spectra in the 0.25-4.0 M concentration range displayed cation-specific behaviors, which enabled the measurement of the appearance concentration thresholds of contact ion pairs between 1.9 and 2.6 M depending on the cation. Conformational explorations performed using a non-local optimization method associated to a polarizable force-field (AMOEBA), followed by high quantum chemistry level (RI-B97-D3/dhf-TZVPP) optimizations, mode-dependent scaled harmonic frequency calculations and electron density analyses, were used to identify the main supramolecular structures contributing to the experimental spectra. A thorough analysis enables us to reveal the mechanisms responsible for the spectroscopic sensitivity of the carboxylate group and the respective role played by the cation and the water molecules, highlighting the necessity of combining advanced experimental and theoretical techniques to provide a fair and accurate description of ion pairing.

A theoretical and experimental case study of the hydrogen bonding predilection of S-methylcysteine.

S-containing amino acids can lead to two types of local NH···S interactions which bridge backbone NH sites to the side chain to form either intra- or inter-residue H-bonds. The present work reports on the conformational preferences of S-methyl-L-cysteine, Cys(Me), using a variety of investigating tools, ranging from quantum chemistry simulations, gas-phase UV and IR laser spectroscopy, and solution state IR and NMR spectroscopies, on model compounds comprising one or two Cys(Me) residues. We demonstrate that in gas phase and in low polarity solution, the C- and N-capped model compound for one Cys(Me) residue adopts a preferred C5-C6γ conformation which combines an intra-residue N-H···O=C backbone interaction (C5) and an inter-residue N-H···S interaction implicating the side-chain sulfur atom (C6γ). In contrast, the dominant conformation of the C- and N-capped model compound featuring two consecutive Cys(Me) residues is a regular type I ß-turn. This structure is incompatible with concomitant C6γ interactions, which are no longer in evidence. Instead, C5γ interactions occur, that are fully consistent with the turn geometry and additionally stabilize the structure. Comparison with the thietane amino acid Attc, which exhibits a rigid cyclic side chain, pinpoints the significance of side chain flexibility for the specific conformational behavior of Cys(Me).

An intraresidue H-bonding motif in selenocysteine and cysteine, revealed by gas phase laser spectroscopy and quantum chemistry calculations.

Models of protein chains containing a seleno-cysteine (Sec) residue have been investigated by gas phase laser spectroscopy in order to document the effect of the H-bonding properties of the SeH group in the folding of the Sec side chain, by comparison with recent data on Ser- and Cys-containing sequences. Experimental data, complemented by quantum chemistry calculations and natural bonding orbital (NBO) analyses, are interpreted in terms of the formation of a so-called 5γ intra-residue motif, which bridges the acceptor chalcogen atom of the side chain to the NH bond of the same residue. This local structure, in which the O/S/Se atom is close to the plane of the N-terminal side amide, is constrained by local backbone-side chain hyperconjugation effects involving the S and Se atoms. Theoretical investigations of the Cys/Sec side chain show that (i) this 5γ motif is an intrinsic feature of these residues, (ii) the corresponding H-bond is strongly non-linear and intrinsically weak, (iii) but enhanced by γ- and ß-turn secondary structures, which promote a more favorable 5γ H-bonding approach and distance. The resulting H-bonds are slightly stronger in selenocysteine than in cysteine, but nearly inexistent in serine, whose side chain in contrast behaves as a H-bonding donor. The modest spectral shifts of the Cys/Sec NH stretches measured experimentally reflect the moderate strength of the 5γ H-bonding, in agreement with the correlation obtained with a NBO-based H-bond strength indicator. The evolution along the Ser, Cys and Sec series emphasizes the compromise between the several factors that control the H-bonding in a hyperconjugation-constrained geometry, among them the chalcogen van der Waals and covalent radii. It also illustrates the 5γ H-bond enhancements with the Sec and Cys residues favoured by the constraints imposed by the γ- and ß-turn structures of the peptide chain.

Intrinsic folding of the cysteine residue: competition between folded and extended forms mediated by the -SH group.

A dual microwave and optical spectroscopic study of a capped cysteine amino acid isolated in a supersonic expansion, combined with quantum chemistry modelling, enabled us to characterize the conformational preferences of Cys embedded in a protein chain. IR/UV double resonance spectroscopy provided evidence for the coexistence of two conformers, assigned to folded and extended backbones (with classical C7 and C5 backbone H-bonding respectively), each of them additionally stabilized by specific main-chain/side-chain H-bonding, where the sulfur atom essentially plays the role of H-bond acceptor. The folded structure was confirmed by microwave spectroscopy, which demonstrated the validity of the DFT-D methods currently used in the field. These structural and spectroscopic results, complemented by a theoretical Natural Bond Orbital analysis, enabled us to document the capacity of the weakly polar -CH2-SH side chain of Cys to adapt itself to the intrinsic local preferences of the peptide backbone, i.e., a γ-turn or a ß-sheet extended secondary structure. The corresponding local H-bonding bridges the side chain acceptor S atom to the backbone NH donor site of the same or the next residue along the chain, through a 5- or a 6-membered ring respectively.

Rationalizing the diversity of amide-amide H-bonding in peptides using the natural bond orbital method.

Natural bond orbital (NBO) analysis of electron delocalization in a series of capped isolated peptides is used to diagnose amide-amide H-bonding and backbone-induced hyperconjugative interactions, and to rationalize their spectral effects. The sum of the stabilization energies corresponding to the interactions between NBOs that are involved in the H-bonding is demonstrated as an insightful indicator for the H-bond strength. It is then used to decouple the effect of the H-bond distance from that, intrinsic, of the donor/acceptor relative orientation, i.e., the geometrical approach. The diversity of the approaches given by the series of peptides studied enables us to illustrate the crucial importance of the approach when the acceptor is a carbonyl group, and emphasizes that efficient approaches can be achieved despite not matching the usual picture of a proton donor directly facing a lone pair of the proton acceptor, i.e., that encountered in intermolecular H-bonds. The study also illustrates the role of backbone flexibility, partly controlled by backbone-amide hyperconjugative interactions, in influencing the equilibrium structures, in particular by frustrating or enhancing the HB for a given geometrical approach. Finally, the presently used NBO-based HB strength indicator enables a fair prediction of the frequency of the proton donor amide NH stretching mode, but this simple picture is blurred by ubiquitous hyperconjugative effects between the backbone and amide groups, whose magnitude can be comparable to that of the weakest H-bonds.

Identification of ion pairs in solution by IR spectroscopy: crucial contributions of gas phase data and simulations.

In a context where structure elucidation of ion pairs in solution remains a contemporary challenge, this work explores an original approach where accurate gas phase spectroscopic data are used to refine high level quantum chemistry calculations of ion pairs in solution, resulting in an unprecedented level of accuracy in vibrational frequency prediction. First, gas phase studies focus on a series of isolated contact ion pairs (M+, Ph-CH2-COO-, with M = Li, Na, K, Rb, Cs) for which conformer-selective IR spectra in the CO2- stretch region are recorded. These experiments reveal the interactions at play in isolated contact ion pairs, and provide vibrational frequencies enabling us to assess the accuracy of the theoretical approach used, i.e., mode-dependent scaled harmonic frequency calculations at the RI-B97-D3/dhf-TZVPP level. This level of calculation is then employed on large water clusters embedding either a free acetate ion or its contact or solvent-shared pairs with a sodium cation in order to simulate the individual vibrational spectra of these species in solution. This study shows that the stretching modes of carboxylate are sensitive to both solvent-shared and contact ion pair formation. FTIR spectra of solutions of increasing concentrations indeed reveal several spectral changes consistent with the presence of specific types of solvent-shared and contact ion pairs. By providing relevant guidelines for the interpretation of solution phase IR spectra, this work illustrates the potential of the approach for the elucidation of supramolecular structures in electrolyte solutions.

Unifying the microscopic picture of His-containing turns: from gas phase model peptides to crystallized proteins.

The presence in crystallized proteins of a local anchoring between the side chain of a His residue, located in the central position of a γ- or ß-turn, and its local main chain environment, was assessed by the comparison of protein structures with relevant isolated model peptides. Gas phase laser spectroscopy, combined with relevant quantum chemistry methods, was used to characterize the γ- and ß-turn structures in these model peptides. A conformer-selective NH stretch infrared study provided evidence for the formation in vacuo of two types of short-range H-bonded motifs, labelled ε-6δ and δ-δ7/πH, bridging the His side chain (in its gauche+ rotamer) to the neighbouring NH(i) and CO(i) sites of the backbone; each side chain-backbone motif was found to be specific of the tautomer (ε or δ) adopted by the His side chain in its neutral form. A close comparison between ß- and γ-turns, selected from the Protein Data Bank, and the gas phase models demonstrated that a significant proportion of the gauche+ His rotamer distribution of proteins was well described by the corresponding gas phase H-bonded structures. This is consistent with the persistence of local 6δ and δ7/πH intramolecular interactions in proteins, emphasizing the relevance of gas phase data to secondary structures that are poorly accessible to solvents, e.g., in the case of a specific compact topology (Xxx-His ß-turns). Deviations from the gas phase structures were also observed, mainly in His-Xxx ß-turns, and assigned to solvent accessible turn structures. They were well accounted for by theoretical models of microhydrated turns, in which a few solvent molecules take over the gas phase motifs, constituting a water-mediated local anchoring of the His side chain to the backbone. Finally, the present gas phase benchmark models also pinpointed weaknesses in the protein structure determination by X-ray diffraction analysis; in particular, besides the lack of tautomer information, inaccuracies in the description of imidazole ring flip rotamerism were identified.

Local NH-π interactions involving aromatic residues of proteins: influence of backbone conformation and ππ* excitation on the π H-bond strength, as revealed from studies of isolated model peptides.

Conformer-selective IR gas phase spectroscopy and high level quantum chemistry methods have been used to characterise the diversity of local NH-π interactions between the π ring of a phenylalanine aromatic residue and the nearby main chain amide groups. The study of model systems shows how the amide NH stretch vibrational features, in the 3410-3460 cm-1 frequency range, can be used to monitor the strength of these local π H-bonds, which is found to depend on both the backbone conformation and the aromatic side chain orientation. This is rationalized in terms of partial electron transfer between the π cloud and the main chain NH bonds, with the help of analysis tools based on Natural Bonding Orbitals and Non-Covalent Interactions plots. The experimental study, extended to the NH-π interactions when the Phe residue is excited in its first ππ* electronic state, also demonstrates the principle of the ππ* labelling technique, i.e. a selective labelling of those NH bonds in a peptide molecule that are in close contact with an aromatic ring, as an elegant tool for IR spectroscopic assignments. The validation of theoretical predictions against experimental data (frequency change upon excitation) eventually qualifies the use of the CC2 method for the description of the ππ* excited states of systems having a phenyl ring, both in terms of structure, vibrational modes and nature of excited states.

Effective Strategy for Conformer-Selective Detection of Short-Lived Excited State Species: Application to the IR Spectroscopy of the N1H Keto Tautomer of Guanine.

The ultrafast deactivation processes in the excited state of biomolecules, such as the most stable tautomers of guanine, forbid any state-of-the-art gas phase spectroscopic studies on these species with nanosecond lasers. This drawback can be overcome by grafting a chromophore having a long-lived excited state to the molecule of interest, allowing thus a mass-selective detection by nanosecond R2PI and therefore double resonance IR/UV conformer-selective spectroscopic studies. The principle is presently demonstrated on the keto form of a modified 9-methylguanine, for which the IR/UV double resonance spectrum in the C═O stretch region, reported for the first time, provides evidence for extensive vibrational couplings within the guanine moiety. Such a successful strategy opens up a route to mass-selective IR/UV spectroscopic investigations on molecules exhibiting natural chromophores having ultrashort-lived excited states, such as DNA bases, their complexes as well as peptides containing short-lived aromatic residues.

Isolated neutral peptides.

This chapter examines the structural characterisation of isolated neutral amino-acids and peptides. After a presentation of the experimental and theoretical state-of-the-art in the field, a review of the major structures and shaping interactions is presented. Special focus is made on conformationally-resolved studies which enable one to go beyond simple structural characterisation; probing flexibility and excited-state photophysics are given as examples of promising future directions.

Intrinsic Folding Proclivities in Cyclic ß-Peptide Building Blocks: Configuration and Heteroatom Effects Analyzed by Conformer-Selective Spectroscopy and Quantum Chemistry.

This work describes the use of conformer-selective laser spectroscopy following supersonic expansion to probe the local folding proclivities of four-membered ring cyclic ß-amino acid building blocks. Emphasis is placed on stereochemical effects as well as on the structural changes induced by the replacement of a carbon atom of the cycle by a nitrogen atom. The amide A IR spectra are obtained and interpreted with the help of quantum chemistry structure calculations. Results provide evidence that the building block with a trans-substituted cyclobutane ring has a predilection to form strong C8 hydrogen bonds. Nitrogen-atom substitution in the ring induces the formation of the hydrazino turn, with a related but distinct hydrogen-bonding network: the structure is best viewed as a bifurcated C8/C5 bond with the N heteroatom lone electron pair playing a significant acceptor role, which supports recent observations on the hydrazino turn structure in solution. Surprisingly, this study shows that the cis-substituted cyclobutane ring derivative also gives rise predominantly to a C8 hydrogen bond, although weaker than in the two former cases, a feature that is not often encountered for this building block.

Intra-residue interactions in proteins: interplay between serine or cysteine side chains and backbone conformations, revealed by laser spectroscopy of isolated model peptides.

Intra-residue interactions play an important role in proteins by influencing local folding of the backbone. Taking advantage of the capability of gas phase experiments to provide relevant information on the intrinsic H-bonding pattern of isolated peptide chains, the intra-residue interactions of serine and cysteine residues, i.e., OH/SH···OC(i) C6 and NH(i···)O/S C5 interactions in Ser/Cys residues, are probed by laser spectroscopy of isolated peptides. The strength of these local side chain-main chain interactions, elegantly documented from their IR spectral features for well-defined conformations of the main chain, demonstrates that a subtle competition exists between the two types of intra-residue bond: the C6 H-bond is the major interaction with Ser, in contrast to Cys where C5 interaction takes over. The restricted number of conformers observed in the gas phase experiment with Ser compared to Cys (where both extended and folded forms are observed) also suggests a significant mediation role of these intra-residue interactions on the competition between the several main chain folding patterns.