Research Activities with Biomolecular Systems

IRMPD Spectroscopy of Biomolecules
Analysis of building blocks of biomolecules: determination of the protonation site
Gas phase structure of flexible peptide : Arginine-Glycine-Aspartic acid (RGD)
Transition between gas-phase and aqueous solution structures of a neurotransmitter
Structural characterization of large biomolecules: DNA strands containing G-quadruplex
Molecular recognition in biomolecular non-covalent complexes
Selected Publications

Laser Desorption on Liquid Microdroplets

Simulation and Theory
Conformational search and predicted IR spectra
Ab-initio excited state calculations
Selected Publications

Ion-Induced Fragmentation of Amino Acids: Effect of the Environment

IRMPD Spectroscopy of Biomolecules

Infrared multiphoton dissociation spectroscopy (IRMPD) is a spectroscopic method, developed in Europe during recent years, which is applicable to a wide class of molecular ions. This technique allows studying the structure of biomolecules in the gas phase through comparison between experimental IR spectra and their prediction from quantum chemistry calculations. Its main advantage is that it is quasi-universal and does not require the presence of a visible/UV chromophore as in R2PI/IR spectroscopy. We investigate ions thermalized at room temperature and produced by electrospray from solutions. Those ions are confined in a quadrupole ion trap mass-spectrometer (Bruker Esquire 3000) and illuminated by the high-power and high-repetition rate infrared beam issued from the CLIO free-electron laser facility (Orsay, France).

In order to interpret experimental IRMPD spectra, two possibilities are offered. The first one consists in a systematic search for the different potential energy minima. IR spectra are subsequently calculated for each low-energy equilibrium conformations. This is usually done within the harmonic approximation with some appropriate scaling factor. Comparison between the experimentally observed spectrum and the predicted spectra is then used to identify populated isomers and/or conformers of the studied system.

An approach closer to experimental conditions consists in simulating the dynamical behaviour of peptides through DFT-based Car-Parrinello molecular dynamics (CPMD) conducted at the experimental temperature. This task is performed in the group of M.P. Gaigeot LAMBE Evry. MD is essential for including conformational dynamics (entropic effects being taken into account without any approximation), and is thus essential for the calculation of IR spectra of complex molecular systems that display numerous energetically equivalent conformations. The main point of IR spectra calculations based on the dipole time correlation function is that all anharmonic effects are naturally taken into account, and no approximations are made. This is to be opposed to the two successive harmonic approximations usually adopted for the determination of IR spectra from static ab initio calculations (harmonic approximation of the potential energy surface at the optimized geometries and mechanical harmonic approximation for the transition dipole moments, as used in normal modes analysis). In particular, the finite temperature dynamics takes place on all accessible parts of the potential energy surface, be they harmonic or anharmonic.

Mass-selected ions absorb photons that bring them from a vi=0 to a vf=1 vibrational state. Between two photon absorptions, internal vibrational redistribution (IVR) takes place and brings back ions to their initial vi=0 vibrational state, while the acquired internal energy is redistributed over other modes. Following absorption of many photons, dissociation takes place. Infrared absorption is then monitored through detection of the ionic fragmentation yield.

Current research topics:

Analysis of building blocks of biomolecules: determination of the protonation site

IRMPD offers a direct possibility for determination of protonation sites by comparison between experimental IR absorption spectrum and the predicted spectra of the different tautomers. In the case of protonated dialanine, static calculations of the lowest energy configurations show that the proton can be located at the N-terninal (labelled A1 and A2) or C=O (labelled O1) amide groups at 300 K. These conformations are separated by energy barriers with transition states accessible at room temperature. Conformations A1 and A2 give the best agreement with the experimental spectrum while conformation O1 contributes to a much less extent to the experimental spectrum.

Car-Parrinello molecular dynamics (CPMD) simulations on Ala-AlaH+ peptide at 300K have shown a continual and recurrent isomerization dynamics between the two A1 and A2 conformers, i.e. the internal energy of the peptide at 300K is sufficient to overcome the energy barrier separating the A1 and A2 basins on the potential energy surface. Besides, one important result is the observation of spontaneous and reversible proton transfer events between NH3+ (A conformer) and the adjacent C=O amide (O conformer). This indicates that O1 is a metastable state which is thermally accessible at 300K. The IR spectrum extracted from the CPMD simulation is calculated from the Fourier Transform of the dipole time correlation function. All pertinent band shaping effects, such as anharmonicity and temperature are incorporated ab inito via the CPMD methodology.

Gas phase structure of flexible peptide : Arginine-Glycine-Aspartic acid (RGD)

An important recognized motif involved in cell adhesion and inflammatory processes is the arginine-glycine-aspartic acid RGD sequence, found in large glycoproteins such as fibronectin, participating to the composition of the intercellular medium. It is specially recognized by transmembrane proteins called integrins and initiates cell-signalling processes.

We have shown that the protonated RGD sequence adopts in the gas phase at least two conformations at 300K, one being similar to the structure of RGD as embedded in a protein (dendroaspin). In this neurotoxin homologue, the flexible RGD loop structure corresponds to residues 43-45 located on the surface of the protein. Our gas-phase study has shown that this loop structure is an intrinsic structural property of RGD, even if the acid aspartic carboxyl group is neutral while it is deprotonated for peptides in solution.

Transition between gas-phase and aqueous solution structures of a neurotransmitter

Molecular recognition of acetylcholine and its agonists by nicotinic or muscarinic acetylcholine receptors is a widely studied systems due to its importance in memory, cognition and reward processes as well as its potential implications in the treatment in Alzheimer's and Parkinson's diseases. Among the key elements for biorecognition of these molecules in the binding sites of the receptors, called "pharmacophores", are a cation-&pi interaction and an accepting hydrogen bond interaction with receptor residues. The distance between these two pharmacophoric sites is thus a crucial parameter that is usually invoked for explaining the strong biochemical similarities observed between acetylcholine and either nicotine and muscarine ions.

Structural information on acetylcholine and its two agonists, nicotine and muscarine has been obtained from the interpretation of IR spectra recorded in the gas-phase (IRMPD) or in low pH aqueous solutions (FT-IR). In the gas-phase, the IRMPD spectrum of acetylcholine ions exhibits three distinct transitions of the carbonyl stretch around 1750 cm-1, which implies that at least three different conformations are populated at 300K. Among these conformers, only one corresponds to a N+-O distance (5.05 Å) that fits within the bioactive range, while the four others low energy conformers exhibit much shorter N+-O distance. However, in aqueous solution, this distance in the four lowest energy conformations is comprised in between 4.6 and 5.2 Å, all compatible with the bioactive range. The conformational space of these very flexible molecules is thus strongly modified by the presence of the solvent.

In the case of nicotine, the solvatation induces a complete change of the structure and the protonation site. Nicotine possesses two protonation sites on the nitrogen of the pyridine (labelled N1(sp2)) and pyrrolidine (labelled N12(sp3)) cycles. In solution, a strong transition at 1450 cm-1 is observed and corresponds to the bending mode of N12-H+. In the gas phase, the IRMPD spectrum of protonated nicotine exhibits an intense transition centred at 1540 cm-1 that involves the bending motion of N1-H+ group. This result has provided for the first time a clear experimental evidence for protonation of nicotine on the pyridine site, which is not the bioactive site of nicotine in condensed phase.

Structural characterization of large biomolecules: DNA strands containing G-quadruplex

Nucleic acid secondary structures are determined by hydrogen bonding interactions between nucleic bases. Besides the well-known Watson-Crick base pairing motif, there is a variety of other possible hydrogen bonding configurations. For instance, guanine-rich sequences can fold into G-quadruplex structures owing to Hoogsteen hydrogen bonding between four guanines, forming G-quartets. As evidence is accumulating on in vivo G-quadruplex formation in telomeric DNA and in the promoters of some oncogenes, these structures have become key targets for anticancer strategies. G-quadruplexes are present in telomeres (ends of human chromosomes) that consist of long DNA single strands containing repeats of TTAGGG sequence.

We have investigated the IR signature of two quadruplex-forming sequences: dTG4T, forming a tetrameric quadruplex [(dTG4T)4(NH4+)3] that is highly stable in solution and in the gas phase, and the human telomeric sequence dTTAGGGTTAGGGTTAGGGTTAGGG (noted T4), forming an intramolecular antiparallel quadruplex in ammonium acetate solution. For both species, a significant red shift of the C=O stretch mode of the guanine base is observed for the non-covalent complexes with ammonium cations sitting in between two successive G-quartets. This first experimental study of large biomolecules has proven the ability of IRMPD spectroscopy for gas-phase conformational analysis of rather complex systems.

Molecular recognition in biomolecular non-covalent complexes

Vancomycin is a naturally occurring glycopeptide antibiotic active against Gram-positive bacteria and is considered as a drug of last resort. Its original use is for the treatment of penicillin-resistant Staphylococcus aureus. Vancomycin binds to the bacteria cell-wall peptidoglycan precursor through noncovalent interactions that inhibit the action of specific bacterial enzymes involved in the development of the cell wall through cross-linking with pentaglycine, leading to its cleavage and finally the death of the bacteria. Early studies have shown that vancomycin binds efficiently to the peptidoglycan precursor UDP-N-acetylmuramyl-LA-DE-LK-DA-DA with a high affinity of 1.6x105 M-1, while further works have demonstrated that the essential region of this peptide responsible for the formation of the complex is the DAlanyl-DAlanine terminus sequence.

The binding of vancomycin to the tripeptide cell-wall precursor analogue Ac2LKDADA involves several intermolecular hydrogen bonds, three with the carboxylate group of the tripeptide and two between amide groups of both species. The binding pocket thus contains the pseudopeptidic chain of vancomycin while the benzoic cycles and the sugar moiety do not seem to be directly involved in the formation of the complex.

The mid-IR spectra (1100-1800 cm-1) of these mass-selected anionic species have been recorded by means of resonant infrared multiphoton dissociation (IRMPD) spectroscopy performed with the free-electron laser CLIO. Structural assignment has been achieved through comparisons with the low-energy conformers obtained from replica-exchange molecular dynamics simulations, for which IR spectra were calculated using a hybrid quantum mechanics/semi-empirical (QM/SE) method at the DFT/B3LYP/6-31+G*/AM1 level. Comparison between deprotonated vancomycin and its non-covalently bound V+Ac2LKDADA complex shows significant spectral shifts of the carboxylate stretches and the Amide I and Amide II modes that are satisfactorily reproduced by the structures known from the condensed phase.

The situation is rather different in the protonated form. Neutralization of the carboxylate group impairs the specific interaction of the deprotonated group of the receptor with vancomycin. Earlier gas-phase studies have suggested that the native structure of the complex might not be preserved in the protonated form, although no direct spectroscopic study has been yet undertaken.

The structure of doubly protonated vancomycin antibiotics with its cell-wall precursor analogue Ac2LKDADA has been investigated in the gas phase through a combined laser spectroscopy, ion mobility (in collaboration with the group of Ph. Dugourd, LASIM) and theoretical modeling approach. Replica-exchange molecular dynamics simulations using the Amber99 force field were performed to explore the potential energy landscape of isolated vancomycin ions, as well as the different binding sites with the receptor. Among the low-energy conformers found, those with a calculated diffusion cross-section consistent with ion mobility experiments were selected for further optimization, and their IR spectra were simulated using a hybrid quantum mechanics/semi empirical (QM/SE) method at the DFT/B3LYP/6-31g(d):AM1 level. Both theoretical and experimental findings provide strong evidence that the native structure of the complex is not preserved in vacuo for the doubly protonated species.

Selected Publications:

Resonant-Infrared Multiphoton Dissociation Spectroscopy of gas-phase protonated peptides. Experiments and Car-Parrinello Dynamics at 300°K.
G. Grégoire, M.P. Gaigeot, D.C. Marinica, J. Lemaire, J.P. Schermann and C. Desfrançois Phys. Chem. Chem. Phys. 9, (24) 3082 (2007).

Experimental observation of the transition between gas-phase and aqueous solution structures for acetylcholine, nicotine and muscarine ions
M. Seydou, G. Grégoire, J. Liquier, J.Lemaire, J.P. Schermann and C. Desfrançois, J. Am. Chem. Soc. 130(12); 4187-4195 (2008)

Infrared Signature of DNA G-quadruplexes in the Gas Phase.
V. Gabelica, F. Rosu, E. De Pauw, J. Lemaire, J.-C. Gillet, J.-C. Poully, F. Lecomte, G. Grégoire, J.-P. Schermann and C. Desfrançois, J. Am. Chem. Soc 130, 1810 (2008)

Probing the specific interactions and structures of gas-phase vancomycin antibiotics with cell-wall precursor through IRMPD spectroscopy
J.-C. Poully, F. Lecomte, N. Nieuwjaer, B. Manil, J. P. Schermann, C. Desfrançois, F. Calvo and G. Grégoire Phys. Chem. Chem. Phys. 12, 3606 (2010)

Combining Ion Mobility Mass Spectrometry and Infrared Multiphoton Dissociation Spectroscopy to probe the structure of gas-phase Vancomycin-Ac2LKDADA non-covalent complex
J.-C. Poully, F. Lecomte, N. Nieuwjaer, B. Manil, J. P. Schermann, C. Desfrançois, G. Grégoire, R. Ballivian, F. Chirot, J. Lemoine, F. Calvo, P. Antoine and Ph. Dugourd. Int. J. Mass. Spectro, 297, 28 (2010)

Laser Desorption on Liquid Microdroplets

We develop an original source producing biomolecules in gas phase. This molecular source is new, does not exist in France and is based on the ultra soft laser desorption from liquid micro droplets under vacuum, as recently proposed in the group of Pr. B. Brutschy (Frankfurt University). The desorbed complexes, initially present in the liquid, will thus be studied in the gas phase under carefully chosen conditions in a controlled environment, close to their native forms in solution. It will allow combining mass spectrometry and laser spectroscopy in order to explore the structural properties of biomolecular complexes.

This project aims to study the specific recognition involved in complexation processes of non-covalently bound systems. One of the unique advantages of this gas phase experiment is to study the local interactions between molecules in a free environment or within a controlled number of water molecules in systems in which the stoichiometry of the drug-receptor complexes is perfectly known.

The liquid droplets (50 µm diam) are generated on demand by a commercial droplet generator (Microdrop) and injected from 300 mbar into a high vacuum chamber (10-5-10-6 mbar) through two differential pumping stages. Upon irradiation by home-made mid-IR broad band laser pulses (centered on an absorption band of the solvent), analyte ions are ejected directly into high vacuum. At this stage, the desorption occurs in between the first stage of a two-field ion optics and the emitted ions are then accelerated and detected by micro channel plates (MCP) located 80 cm downstream in a second vacuum chamber.

We are currently building a reflectron-type TOF mass spectrometer that will be installed in the second vacuum chamber. In that configuration, the ions will pass through decelerating ion optics and will be spatially focussed in the extraction region of the orthogonal TOF mass spectrometer. The reflector assembly should ensure a good mass resolution. The mass-spectrometer vacuum chamber will be equipped with several laser feedthroughs before and into the reflector zone to perform laser photodissociation spectroscopy.

Simulation and Theory

We possess a 48-processors PC cluster (Alineos, AMD 2.5 GHz for a total of 96 GB of RAM, dual Gbit switch connecting 18 nodes) suitable to perform high level calculations (running REMD, Turbomole and Gaussian parallelized codes) on rather large molecular systems.

Conformational search and predicted IR spectra:

The structural assignment of biomolecules studying by means of IR spectroscopy is done by comparison with high level ab initio calculation on low lying isomers. The theoretical treatment can be done in our Laboratory and through collaborations with theoretical groups with whom we already worked with:
-M.P. Gaigeot (LAMBE Evry) for Car-Parrinello Molecular Dynamics simulations,
-F. Calvo (LASIM Lyon) for exhaustive exploration of the potential energy surface through Replica Exchange Molecular Dynamics (REMD) simulations using Amber force field.

We have recently evaluated several theoretical methods (DFT, DFT-D, MP2 and hybrid QM/MM at the B3LYP/AM1 level) for assignment of IR spectra of gas phase biomolecules, including nucleobases, amino acids, peptides, sugars, neurotransmitters and antibiotics. Depending on the size of molecular species, these methods can be applied accordingly with a prediction error of the IR transition positions ranging roughly in between 10 and 15 cm-1 even for large molecular systems presently under investigation.

Ab-initio excited state calculations:

Excitation energies (vertical and adiabatic) and response properties are calculated with the CC2 method, which is a simplified and cost-effective variant of the coupled-cluster method with single and double excitations. These calculations are carried out with the TURBOMOLE suite of program, making use of the resolution-of-the-identity (RI) approximation for the evaluation of the electron-repulsion integrals

Selected Publications:

Ab initio study of the excited state deactivation pathways of protonated Tryptophan and Tyrosine.
G. Grégoire, C. Jouvet, C. Dedonder-Lardeux, A. Sobolewski, J. Am. Chem. Soc. 129, 6223 (2007).

Ab-initio molecular dynamics of protonated dialanine and comparison to infrared multiphoton dissociation experiments.
D.C. Marinica, G. Grégoire, C. Desfrançois, J.P. Schermann, D. Borgis and M.P. Gaigeot, J. Phys. Chem. A 110, 8802 (2006).

Transferable Specific Scaling Factors for Interpretation of Infrared Spectra of Biomolecules from Density Functional Theory
Y. Bouteiller, J.C. Gillet, G. Gregoire, J.P. Schermann, J. Phys. Chem. A 112, 11656 (2008)

Evaluation of MP2, DFT, and DFT-D Methods for the Prediction of Infrared Spectra of Peptides
Y. Bouteiller, J.C. Poully, C. Desfrançois, G. Gregoire, J. Phys. Chem. A 113, 630 (2009)

Evaluation of the ONIOM method for interpretation of infrared spectra of gas-phase molecules of biological interest
J.C. Poully, G. Gregoire, J.P. Schermann, J. Phys. Chem. A 113, 8020 (2009)

Ion-Induced Fragmentation of Amino Acids: Effect of the Environment

Chromosomes of eukaryotic cells consist of about the same amounts of DNA and histone proteins. Being the building blocks of these proteins, amino acids belong to the repertoire of molecules relevant in the context of biological radiation damage. Detailed knowledge about molecular mechanisms underlying this damage is of prime importance, for example, in the context of radiotherapy of tumours, but also to improve our fundamental understanding of biological radiation damage in general. Ion-induced radiation damage of biomolecular systems could elucidate the primary processes in hadron therapy; an emerging cancer treatment method. Another aspect of the irradiation studies of amino acids is linked to prebiotic molecules. When considering the scenario of an exogenous delivery of these molecules on planets, it is important to investigate biomolecular responses to all types of radiation present in the interstellar medium.

At the ARIBE Facility (Caen, France), with the groups of B.A. Huber (Caen) and T. Schalhölter (KVI, Groningen, Netherlands), we studied the interaction of 300 keV Xe20+ ions with two amino acids: [D2]glycine (NH2CD2COOH) and valine [NH2(CH)2(CH3)2COOH]. For the isolated molecule, the use of an incident projectile with a higher charge state has the advantage of being a soft ionisation tool due to gentle interactions at large distances. Thus, a smaller amount of energy is transferred, leaving the system in a less-excited state. An estimate of the internal energy of the fragmenting systems, obtained by applying the simplified model of an evaporative ensemble to the glycine molecule, yields values between 5 and 6.5 eV depending on the model parameters. To identify the effect of the environment, we extend the study to clusters of glycine and valine. The fragmentation patterns of the molecules isolated and embedded in clusters can be compared and the fragmentation dynamics discussed.

In the case of isolated amino acids in the gas phase, ion irradiation provokes a unimolecular dissociation process, in particular, the fragmentation is driven by the cleavage of the C-Cα bond, which appears to be the weakest bond of the system. For the larger valine molecule, the loss of the side chain appears as an alternative dissociation process. The valine cation is found to be unstable on the µs timescale.

In the case of amino acid clusters, the first striking result is the observation of the amino acid cation, which is totally absent for the isolated valine molecule and strongly enhanced in the case of glycine. The kinetic energy of these cations indicates that they result from the fragmentation of larger clusters cooling the system. This buffer effect of the cluster is also evidenced by the fact that the fragmentation yield is strongly reduced. The signal intensities associated with the main fragmentation channels for the C-Cα bond cleavage are reduced, which confirms that the amino acid clusters dissipate their excess energy by the evaporation of monomers or by cluster fission. The fragmentation pattern, that is, the possible pathways are also modified. In particular, the decay leading to the formation of the COOH+ ions is quenched in the case of both glycine and valine clusters. The disappearance of this fragmentation channel can be explained by a rapid charge redistribution within the cluster.

Related publication: P. Rousseau et al., ChemPhysChem 12, 930-936 (2011)