Variable-Temperature Tip-Enhanced Raman Spectroscopy of Single-Molecule Fluctuations and Dynamics.

Structure, dynamics, and coupling involving single-molecules determine function in catalytic, electronic or biological systems. While vibrational spectroscopy provides insight into molecular structure, rapid fluctuations blur the molecular trajectory even in single-molecule spectroscopy, analogous to spatial averaging in measuring large ensembles. To gain insight into intramolecular coupling, substrate coupling, and dynamic processes, we use tip-enhanced Raman spectroscopy (TERS) at variable and cryogenic temperatures, to slow and control the motion of a single molecule. We resolve intrinsic line widths of individual normal modes, allowing detailed and quantitative investigation of the vibrational modes. From temperature dependent line narrowing and splitting, we quantify ultrafast vibrational dephasing, intramolecular coupling, and conformational heterogeneity. Through statistical correlation analysis of fluctuations of individual modes, we observe rotational motion and spectral fluctuations of the molecule. This work demonstrates single-molecule vibrational spectroscopy beyond chemical identification, opening the possibility for a complete picture of molecular motion ranging from femtoseconds to minutes.

Molecular simulation-based structural prediction of protein complexes in mass spectrometry: the human insulin dimer.

Protein electrospray ionization (ESI) mass spectrometry (MS)-based techniques are widely used to provide insight into structural proteomics under the assumption that non-covalent protein complexes being transferred into the gas phase preserve basically the same intermolecular interactions as in solution. Here we investigate the applicability of this assumption by extending our previous structural prediction protocol for single proteins in ESI-MS to protein complexes. We apply our protocol to the human insulin dimer (hIns2) as a test case. Our calculations reproduce the main charge and the collision cross section (CCS) measured in ESI-MS experiments. Molecular dynamics simulations for 0.075 ms show that the complex maximizes intermolecular non-bonded interactions relative to the structure in water, without affecting the cross section. The overall gas-phase structure of hIns2 does exhibit differences with the one in aqueous solution, not inferable from a comparison with calculated CCS. Hence, care should be exerted when interpreting ESI-MS proteomics data based solely on NMR and/or X-ray structural information.

Structure and dynamics of oligonucleotides in the gas phase.

By combining ion-mobility mass spectrometry experiments with sub-millisecond classical and ab initio molecular dynamics we fully characterized, for the first time, the dynamic ensemble of a model nucleic acid in the gas phase under electrospray ionization conditions. The studied oligonucleotide unfolds upon vaporization, loses memory of the solution structure, and explores true gas-phase conformational space. Contrary to our original expectations, the oligonucleotide shows very rich dynamics in three different timescales (multi-picosecond, nanosecond, and sub-millisecond). The shorter timescale dynamics has a quantum mechanical nature and leads to changes in the covalent structure, whereas the other two are of classical origin. Overall, this study suggests that a re-evaluation on our view of the physics of nucleic acids upon vaporization is needed.

Real-time observation of the formation of excited radical ions in bimolecular photoinduced charge separation: absence of the Marcus inverted region explained.

Unambiguous evidence for the formation of excited ions upon ultrafast bimolecular photoinduced charge separation is found using a combination of femtosecond time-resolved fluorescence up-conversion, infrared and visible transient absorption spectroscopy. The reaction pathways are tracked by monitoring the vibrational energy redistribution in the product after charge separation and subsequent charge recombination. For moderately exergonic reactions, both donor and acceptor are found to be vibrationally hot, pointing to an even redistribution of the energy dissipated upon charge separation and recombination in both reaction partners. For highly exergonic reactions, the donor is very hot, whereas the acceptor is mostly cold. The asymmetric energy redistribution is due to the formation of the donor cation in an electronic excited state upon charge separation, confirming one of the hypotheses for the absence of the Marcus inverted region in photoinduced bimolecular charge separation processes.

Structural prediction of a rhodamine-based biosensor and comparison with biophysical data.

The predicted structure has been calculated for a protein-based biosensor for inorganic phosphate (Pi), previously developed by some of us (Okoh et al., Biochemistry, 2006, 45, 14764). This is the phosphate binding protein from Escherichia coli labelled with two rhodamine fluorophores. Classical molecular dynamics and hybrid Car-Parrinello/molecular mechanics simulations allow us to provide molecular models of the biosensor both in the presence and in the absence of Pi. In the latter case, the rhodamine fluorophores maintain a stacked conformation in a 'face A to face B' orientation, which is different from the 'face A to face A' stacked orientation of free fluorophores in aqueous solution (Ilich et al., Spectrochim. Acta, Part A, 1996, 52, 1323). A protein conformation change upon binding Pi prevents significant stacking of the two rhodamines. In both states, the rhodamine fluorophores form hydrophobic contact with LEU291, without establishing significant hydrogen bonds with the protein. The accuracy of the models is established by a comparison between calculated and experimental absorption and circular dichroism spectra.

On the role of hydrogen bonds in photoinduced electron-transfer dynamics between 9-fluorenone and amine solvents.

Using ultrafast fluorescence upconversion and mid-infrared spectroscopy, we explore the role of hydrogen bonds in the photoinduced electron transfer (ET) between 9-fluorenone (FLU) and the solvents trimethylamine (TEA) and dimethylamine (DEA). FLU shows hydrogen-bond dynamics in the methanol solvent upon photoexcitation, and similar effects may be anticipated when using DEA, whereas no hydrogen bonds can occur in TEA. Photoexcitation of the electron-acceptor dye molecule FLU with a 400 nm pump pulse induces ultrafast ET from the amine solvents, which is followed by 100 fs IR probe pulses as well as fluorescence upconversion, monitoring the time evolution of marker bands of the FLU S(1) state and the FLU radical anion, and an overtone band of the amine solvent, marking the transient generation of the amine radical cation. A comparison of the experimentally determined forward charge-separation and backward charge-recombination rates for the FLU-TEA and FLU-DEA reaction systems with the driving-force dependencies calculated for the forward and backward ET rates reveals that additional degrees of freedom determine the ET reaction dynamics for the FLU-DEA system. We suggest that hydrogen bonding between the DEA molecules plays a key role in this behaviour.

The hydrogen-bonded 2-pyridone dimer model system. 2. Femtosecond mid-infrared pump-probe study.

2-Pyridone (PD) tautomerises to 2-hydroxypyridine (HP) in liquid solution, the equilibrium of which is solvent dependent. Dimerization of PD and HP leads to the cyclic dimers (PD)(2), (HP)(2), and (PD-HP). A combined NMR and FT-IR study [Szyc, L.; et al. J. Phys. Chem. A 2010, 114, 7749-7760] has shown that solutions of 2-pyridone in CD(2)Cl(2) constitute mainly PD-CD(2)Cl(2) solute-solvent complexes and cyclic dimers (PD)(2). Because of a lack of specific marker modes, a contribution of the cyclic dimer (HP)(2) to the NH/OH stretching absorption between 2400 and 3300 cm(-1) could not be fully ruled out. Here, we present the first ultrafast infrared (IR) pump-probe experiments on the NH/OH stretching region of a solution of 2-pyridone in CD(2)Cl(2). The temporally and spectrally resolved data reveal different rate-like relaxation processes with time constants between 150 fs and 20 ps as well as coherent low-frequency oscillations due to hydrogen bond modes. An analysis shows that the transient behavior is dominated by a single hydrogen bonded species. We compare the low-frequency wavepacket motions, observed with 99 and 150 cm(-1) frequencies, with literature values as well as our quantum chemical calculations and conclude that this single molecular species is cyclic (PD)(2).

The hydrogen-bonded 2-pyridone dimer model system. 1. Combined NMR and FT-IR spectroscopy study.

2-Pyridone (PD), converting to 2-hydroxypyridine (HP) through a lactam-lactim isomerization mechanism, can form three different cyclic dimers by hydrogen bond formation: (PD)(2), (PD-HP), and (HP)(2). We investigate the complexation chemistry of pyridone in dichloromethane-d(2) using a combined NMR and Fourier transform infrared (FT-IR) approach. Temperature-dependent (1)H NMR spectra indicate that at low temperatures (<200 K) pyridone in solution predominantly exists as a cyclic (PD)(2) dimer, in exchange with PD monomers. At higher temperatures a proton exchange mechanism sets in, leading to a collapse of the doublet of (15)N labeled 2-pyridone. Linear FT-IR spectra indicate the existence of several pyridone species, where, however, a straightforward interpretation is hampered by extensive spectral overlap of many vibrational transitions in both the fingerprint and the NH/OH stretching regions. Two-dimensional IR correlation spectroscopy applied on concentration-dependent and temperature-dependent data sets reveals the existence of the (PD)(2) cyclic dimer, of PD-CD(2)Cl(2) solute-solvent complexes, and of PD-PD chainlike dimers. Regarding the difference in effective time scales of the NMR and FT-IR experiments, milliseconds vs (sub)picoseconds, the cyclic dimers (PD-HP) and (HP)(2), and the chainlike conformations HP-PD, may function as intermediates in reaction pathways through which the protons exchange between PD units in cyclic (PD)(2).

Concerted electron and proton transfer in ionic crystals mapped by femtosecond x-ray powder diffraction.

X-ray powder diffraction, a fundamental technique of structure research in physics, chemistry, and biology, is extended into the femtosecond time domain of atomic motions. This allows for mapping (macro)molecular structure generated by basic chemical and biological processes and for deriving transient electronic charge density maps. In the experiments, the transient intensity and angular positions of up to 20 Debye Scherrer reflections from a polycrystalline powder are measured and atomic positions and charge density maps are determined with a combined spatial and temporal resolutions of 30 pm and 100 fs. We present evidence for the so far unknown concerted transfer of electrons and protons in a prototype material, the hydrogen-bonded ionic ammonium sulfate [(NH(4))(2)SO(4)]. Photoexcitation of ammonium sulfate induces a sub-100 fs electron transfer from the sulfate groups into a highly confined electron channel along the c-axis of the unit cell. The latter geometry is stabilized by transferring protons from the adjacent ammonium groups into the channel. Time-dependent charge density maps derived from the diffraction data display a periodic modulation of the channel's charge density by low-frequency lattice motions with a concerted electron and proton motion between the channel and the initial proton binding site. Our results set the stage for femtosecond structure studies in a wide class of (bio)molecular materials.

NMR Studies of Hetero-Association of Caffeine with di-O-Caffeoylquinic Acid Isomers in Aqueous Solution.

Caffeine hetero-association with 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid in aqueous solution has been investigated by one-dimensional (1D) and two-dimensional (2D) high resolution 1H and 13C NMR spectroscopy. Self-association of the di-O-caffeoylquinic acid isomers has been studied as well. Caffeine-di-O-caffeoylquinic acid isomers association constants were measured. The value of the association constant of the caffeine-di-O-caffeoylquinic acid complexes is compatible with previous studies and within the typical range of reported association constants for other caffeine-polyphenols complexes. Structural features of the three different complexes have also been investigated by NMR spectroscopy combined with quantum chemical calculations, and the complex conformation is discussed. Our results show that stacking interactions drive the formation of the complexes and that multiple equilibria are present in the interaction of caffeine with 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid while the complex with 3,5-di-O-caffeoylquinic acid seems to be better defined.

Developing predictive rules for coordination geometry from visible circular dichroism of copper(II) and nickel(II) ions in histidine and amide main-chain complexes.

Circular dichroism (CD) spectroscopy in the visible region (vis-CD) is a powerful technique to study metal-protein interactions. It can resolve individual d-d electronic transitions as separate bands and is particularly sensitive to the chiral environment of the transition metals. Modern quantum chemical methods enable CD spectra calculations from which, along with direct comparison with the experimental CD data, the conformations and the stereochemistry of the metal-protein complexes can be assigned. However, a clear understanding of the observed spectra and the molecular configuration is largely lacking. In this study, we compare the experimental and computed vis-CD spectra of Cu(2+)-loaded model peptides in square-planar complexes. We find that the spectra can readily discriminate the coordination pattern of Cu(2+) bound exclusively to main-chain amides from that involving both main-chain amides and a side-chain (i.e. histidine side-chain). Based on the results, we develop a set of empirical rules that relates the appearance of particular vis-CD spectral features to the conformation of the complex. These rules can be used to gain insight into coordination geometries of other Cu(2+)- or Ni(2+)-protein complexes.

Role of the Membrane Dipole Potential for Proton Transport in Gramicidin A Embedded in a DMPC Bilayer.

The membrane potential at the water/phospholipid interfaces may play a key role for proton conduction of gramicidin A (gA). Here we address this issue by Density Functional Theory-based molecular dynamics and metadynamics simulations. The calculations, performed on gA embedded in a solvated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membrane environment (about 2,000 atoms), indicate that (i) the membrane dipole potential rises at the channel mouth by ∼0.4 V. A similar value has been measured for gA embedded in a DMPC monolayer; (ii) the calculated free energy barrier is located at the channel entrance, consistent with experiments comparing gA proton conduction in different bilayers. The electronic structures of the proton ligands (water molecules and peptide units) are similar to those in the bulk solvent. Based on these results, we suggest an important role of the membrane dipole potential for the free energy barrier of proton permeation of gA. This may provide a rationale for the large increase in the rate of proton conduction under application of a transmembrane voltage, as observed experimentally. Our calculations might suggest also a role for proton desolvation for the permeation process. This role has already emerged from EVB calculations on gA embedded in a model membrane.

Density functional study of Cu(2+)-phenylalanine complex under micro-solvation environment.

We present an atomistic study carried out using density functional calculations including structural relaxations and Car-Parrinello Molecular Dynamics (CPMD) simulations, aiming to investigate the structures of phenylalanine-copper (II) ([Phe-Cu](2+)) complexes and their micro-solvation processes. The structures of the [Phe-Cu](2+) complex with up to four water molecules are optimized using the B3LYP/6-311++G** model in gas phase to identify the lowest energy structures at each degree of solvation (n=0-4). It is found that the phenylalanine appears to be in the neutral form in isolated and mono-hydrated complexes, but in the zwitterionic form in other hydrated complexes (with n≥2). The most stable structures of the complexes suggest that the Cu(2+)-π interactions are not dominant in the [Phe-Cu](2+) complexes. The present CPMD simulations of the lowest energy micro-hydrated [Phe-Cu](2+) complexes also reveal that the maximum coordination of Cu(2+) in the presence of the Phe ligand does not exceed four: the oxygen atoms from three water molecules and one carboxyl oxygen atom of Phe. Any excess water molecules will migrate to the second solvation shell. Moreover a unique structural motif, (N)H···O(3)···H2O-Cu(2+) is present in the lowest energy complexes, which is recognized to be significant in stabilizing the structures of the complexes. Extensively rich information of the structures, energetics, hydrogen bonds and dynamics of the lowest energy complexes are discussed.

Ion permeation in the NanC porin from Escherichia coli: free energy calculations along pathways identified by coarse-grain simulations.

Using the X-ray structure of a recently discovered bacterial protein, the N-acetylneuraminic acid-inducible channel (NanC), we investigate computationally K(+) and Cl(-) ions' permeation. We identify ion permeation pathways that are likely to be populated using coarse-grain Monte Carlo simulations. Next, we use these pathways as reaction coordinates for umbrella sampling-based free energy simulations. We find distinct tubelike pathways connecting specific binding sites for K(+) and, more pronounced, for Cl(-) ions. Both ions permeate the porin preserving almost all of their first hydration shell. The calculated free energy barriers are G(#) ≈ 4 kJ/mol and G(#) ≈ 8 kJ/mol for Cl(-) and K(+), respectively. Within the approximations associated with these values, discussed in detail in this work, we suggest that the porin is slightly selective for Cl(-) versus K(+). Our suggestion is consistent with the experimentally observed weak Cl(-) over K(+) selectivity. A rationale for the latter is suggested by a comparison with previous calculations on strongly anion selective porins.

Unraveling the structure of hydrogen bond stretching mode infrared absorption bands: an anharmonic density functional theory study on 7-azaindole dimers.

The structure of the linear infrared absorption spectrum of the N-H stretching mode in 7-azaindole dimers is analyzed by quartic anharmonic vibrational force field calculations based on density functional theory. It is demonstrated that a multiple Fermi resonance model including contributions from 12 fingerprint vibrational modes, most of them containing considerable contributions of N-H bending motions, combined with a single low-frequency mode satisfactorily explains the complex line shape of N-H stretching mode absorption band.

Mode-selective O-H stretching relaxation in a hydrogen bond studied by ultrafast vibrational spectroscopy.

The ultrafast relaxation of the excited O-H stretching vibration is studied by ultrafast infrared-pump/infrared-probe and infrared-pump/Raman-probe spectroscopy. We demonstrate a 200 fs lifetime of the hydrogen-bonded O-H stretching mode in 2-(2'-hydroxy-5'-methyl-phenyl)benzotriazole (TINUVIN P). O-H stretching relaxation occurs through a few major channels that all involve combination and overtone bands of modes with considerable in-plane O-H bending character. In particular, the mode, which contains the largest O-H bending contribution, plays a prominent role for primary processes of intramolecular vibrational energy redistribution. Theoretical calculations of vibrational energy transfer rates based on a Fermi golden rule approach account for the experimental findings.

Hydrogen-bonded acetic acid dimers: anharmonic coupling and linear infrared spectra studied with density-functional theory.

Anharmonic vibrational force field calculations provide a quantitative understanding of the width and substructure of the linear IR-absorption spectrum of the O-H stretching mode in acetic acid dimers (CH3-COOH)2 and (CD3-COOH)2. Anharmonic coupling of the high-frequency upsilon(OH) mode to fingerprint and low-frequency modes is included resulting in 11- and 9-dimensional vibrational Hamiltonians. A sixth-order force field covering up to three-body interactions is used. Force constants are calculated by fitting one-dimensional potential-energy surfaces and a finite difference procedure applying density-functional theory [Becke 3 Lee-Yang-Parr 6-311+G(d,p)]. It is demonstrated that both anharmonic coupling to low-frequency modes as well as Fermi resonance coupling with fingerprint modes are important mechanisms explaining the line shape of the O-H stretching IR-absorption band in acetic acid dimers.

Solvent-dependent photoacidity state of pyranine monitored by transient mid-infrared spectroscopy.

We investigate with femtosecond mid-infrared spectroscopy the vibrational-mode characteristics of the electronic states involved in the excited-state dynamics of pyranine (HPTS) that ultimately lead to efficient proton (deuteron) transfer in H2O (D2O). We also study the methoxy derivative of pyranine (MPTS), which is similar in electronic structure but does not have the photoacidity property. We compare the observed vibrational band patterns of MPTS and HPTS after electronic excitation in the solvents: deuterated dimethylsulfoxide, deuterated methanol and H2O/D2O, from which we conclude that for MPTS and HPTS photoacids the first excited singlet state appears to have charge-transfer (CT) properties in water within our time resolution (150 fs), whereas in aprotic dimethylsulfoxide the photoacid appears to be in a non-polar electronic excited state, and in methanol (less polar and less acidic than water) the behaviour is intermediate between these two extremes. For the fingerprint vibrations we do not observe dynamics on a time scale of a few picoseconds, and with our results obtained on the O-H stretching vibration we argue that the dynamic behaviour observed in previous UV/Vis pump-probe studies is likely to be related to solvation dynamics.

Sequential proton transfer through water bridges in acid-base reactions.

The proton transfer mechanism between aqueous Brønsted acids and bases, forming an encounter pair, has been studied in real time with ultrafast infrared spectroscopy. The transient intermediacy of a hydrated proton, formed by ultrafast dissociation from an optically triggered photoacid proton donor ROH, is implicated by the appearance of an infrared absorption marker band before protonation of the base, B-. Thus, proton exchange between an acid and a base in aqueous solution is shown to proceed by a sequential, von Grotthuss-type, proton-hopping mechanism through water bridges. The spectra suggest a hydronium cation H3O+ structure for the intermediate, stabilized in the Eigen configuration in the ionic complex RO-...H3O+...B-.