Vibrational Probe Research Articles

Carbon−Deuterium Vibrational Probes of Amino Acid Protonation State

The protonation state of titratable amino acid residues has profound effects on protein stability and function. Therefore, correctly determining the acid dissociation constant, pK(a), of charged residues under physiological conditions is an important challenge. The general utility of site-specific carbon-deuterium (C-D) vibrational probes as reporters of the protonation state of arginine, aspartic acid, glutamic acid, and lysine amino acid side chains was examined using density functional theory (DFT) calculations. Substantial shifts were observed in the anharmonic vibrational frequencies of a C-D(2) probe placed immediately adjacent to the titratable group. Lysine exhibited the largest C-D(2) frequency shifts upon protonation, 44.9 cm(-1) (symmetric stretch) and 69.5 cm(-1) (asymmetric stretch). Furthermore, the predicted harmonic intensities of the C-D(2) probe vibrations were extraordinarily sensitive to the protonation state of the nearby acidic or basic group. Accounting for this dramatic change in intensity is essential to the interpretation of an infrared (IR) absorption spectrum that contains the signature of both the neutral and charged states.

Cyanylated Cysteine Is a Site-Specific Vibrational Probe of Disorder-to-Order Transitions In Helical Protein Domains

The time scale of molecular vibrations allows infrared spectroscopy to be a picosecond probe of fluctuations in the local solvent and the structural environment of distinct vibrations. Solvent-exposed, free cysteine side chains are easily modified to thiocyanate through established reaction chemistry. Following site-directed mutagenesis to introduce cysteine, this modification allows the site-specific placement of thiocyanate in disordered domains implicated in binding or other structure-inducing events. Using a natural system (the Ntail protein from measles virus) and model helical peptides, we demonstrate that the frequency and line shape of the CN stretching band of cyanylated cysteine are sensitive to formation of both secondary structure and tertiary/quaternary or lipid contacts. The CN line shape indicates significant attenuation of the dynamics of water surrounding well-formed secondary structures.

Multiple Anharmonic Vibrational Probes of Sugar Structure and Dynamics

Quantum mechanical computations and molecular dynamics simulations are carried out for the simplest sugar glycolaldehyde to gain insight into the underlying force fields that determine the vibrational spectroscopic parameters relevant to structure and dynamics. The harmonic and anharmonic vibrational frequencies of the 3N--6 modes and their diagonal and off-diagonal anharmonicities are evaluated using the hybrid B3LYP functional in comparison with other high-level theories. Very good performance of B3LYP/6-31+G** is found in predicting the anharmonic frequencies by statistical analysis and by comparison to gas-phase experiments. Full cubic and semidiagonal quartic anharmonic force constants, the origin of the anharmonicities, the isotope dependence of the anharmonicities, and the polarizable continuum solvent effect on the anharmonicities are examined, in particular, for the CO, C-H, and O-H stretching modes. Site-dependent dynamical interactions between glycolaldehyde and water molecules in the hydration shells are examined by molecular dynamics simulations employing a set of molecular mechanical force fields developed on the basis of quantum mechanical computations. The statistical distributions and correlations of the fundamental transition frequencies and transition dipoles are obtained through instantaneous normal-mode analysis. The simultaneous assessment of multiple parameters of multiple vibrational probes shall prove useful in understanding the characteristics of sugar structure and dynamics expressed in two-dimensional infrared correlation spectra.

Nitrile groups as vibrational probes of biomolecular structure and dynamics: an overview

This paper presents an overview of recent experiments and theoretical developments aimed at using vibrational spectroscopy to understand the structure and dynamics of nitrile-labeled biomolecules. Nitrile groups are excellent vibrational probes of proteins and DNA because they absorb in a region of the spectrum that is relatively free of absorption due to the biomolecule, and they have high extinction coefficients. The vibrational frequency of nitrile groups is also extraordinarily sensitive to its local environment, and thus C[triple bond, length as m-dash]N bonds have been employed in both linear and 2-D infrared (IR) spectroscopy experiments and also as vibrational Stark probes of electric fields in proteins. The interpretation and design of these experiments would be enhanced by accurate calculations of IR spectra from molecular dynamics simulations. Recently, theoretical developments towards computing the vibrational spectrum of nitrile groups in the condensed-phase have been highly successful. A strong synergy between experiment and theory will further promote the use of vibrational spectroscopy of nitrile-labeled biomolecules to address fundamental questions of structure and dynamics that are elusive to other techniques.

A Vibrational Probe for Local Nucleic Acid Environments: 5-Cyano-2′-deoxyuridine

Nitriles have been shown to be effective vibrational probes of local environments in proteins but have yet to be fully utilized for the study of nucleic acids. The potential utility of 5-cyano-2'-deoxyuridine ( 1) as a probe of local nucleic acid environment was investigated by measuring the dependence of the IR nitrile stretching frequency (nu CN), line shape, and absorbance on solvent and temperature. The nu CN was found to be sensitive to solvent with an observed blue shift of 9.2 cm (-1) in going from THF to water. The dependence of the nitrile IR absorbance band was further investigated in water-THF mixtures. Global line shape analysis, difference FTIR spectroscopy, and singular value decomposition (SVD) were used to show the presence of three distinct local environments around the nitrile group of 1 in these mixtures. A modest blue shift in nu CN was observed upon a hydrogen-bond-mediated heterodimer formation between 2 (a silyl ether analogue of 1) and 2,6-diheptanamido-pyridine ( 3a) in chloroform. The intrinsic temperature dependence of the nu CN was found to be minimal and linear over the temperature range studied. The experimental studies were complemented by density functional theory (DFT) calculations on the dependence of the nitrile stretching frequency on solute-solvent interactions and upon heterodimer formation with model systems.

Reaction Dynamics and Vibrational Spectroscopy of CH3D Molecules with Both C−H and C−D Stretches Excited

State-resolved reactions of CH3D molecules containing both C-H and C-D stretching excitation with Cl atoms provide new vibrational spectroscopy and probe the consumption and disposal of vibrational energy in the reactions. The vibrational action spectra have three different components, the combination of the C-H symmetric stretch and the C-D stretch (nu1 + nu2), the combination of the C-D stretch and the C-H antisymmetric stretch (nu2 + nu4), and the combination of the C-D stretch and the first overtone of the CH3 bend (nu2 + 2nu5). The simulation for the previously unanalyzed (nu2 + nu4) state yields a band center of nu0 = 5215.3 cm(-1), rotational constants of A = 5.223 cm(-1) and B = 3.803 cm(-1), and a Coriolis coupling constant of zeta = 0.084. The reaction dynamics largely follow a spectator picture in which the surviving bond retains its initial vibrational excitation. In at least 80% of the reactive encounters of vibrationally excited CH3D with Cl, cleavage of the C-H bond produces CH2D radicals with an excited C-D stretch, and cleavage of the C-D bond produces CH3 radicals with an excited C-H stretch. Deviations from the spectator picture seem to reflect mixing in the initially prepared eigenstates and, possibly, collisional coupling during the reaction.

Investigation of an unnatural amino acid for use as a resonance Raman probe: Detection limits, solvent and temperature dependence of the νC≡N band of 4-cyanophenylalanine.

The incorporation of unnatural amino acids into proteins that act as spectroscopic probes can be used to study protein structure and function. One such probe is 4-cyanophenylalanine (PheCN), the nitrile group of which has a stretching mode that occurs in a region of the vibrational spectrum that does not contain any modes from the usual components of proteins and the wavenumber is sensitive to the polarity of its environment. In this work we evaluate the potential of UV resonance Raman spectroscopy for monitoring the sensitivity of the νC≡N band of PheCN incorporated into proteins to the protein environment. Measurement of the Raman excitation profile of PheCN showed that considerable resonance enhancement of the Raman signal was obtained using UV excitation and the best signal-to-noise ratios were obtained with excitation wavelengths of 229 and 244 nm. The detection limit for PheCN in proteins was ~10 μM, approximately a hundred-fold lower than the concentrations used in IR studies, which increases the potential applications of PheCN as a vibrational probe. The wavenumber of the PheCN νC≡N band was strongly dependent on the polarity of its environment, when the solvent was changed from H(2)O to THF it decreased by 8 cm(-1). The presence of liposomes caused a similar though smaller decrease in νC≡N for a peptide, mastoparan X, modified to contain PheCN. The selectivity and sensitivity of resonance Raman spectroscopy of PheCN mean that it can be a useful probe of intra- and intermolecular interactions in proteins and opens the door to its application in the study of protein dynamics using time-resolved resonance Raman spectroscopy.

Infrared absorption line shapes in the classical limit: A comparison of the classical dipole and fluctuating frequency approximations

Infrared spectroscopy is a versatile technique for probing the structure and dynamics of condensed-phase systems. Simulating infrared absorption spectra with molecular dynamics (MD) offers a powerful means to establish a molecular-level interpretation of experimental results, as well as a basis for the parametrization of more accurate simulation force-fields. Two distinct methods for the calculation of infrared absorption line shapes of high-frequency (Planck's omega/k(B)T>>1) vibrational probes from MD simulations are examined: The classical dipole approximation (CDA) and the fluctuating frequency approximation (FFA). Although these two formalisms result in expressions for the infrared absorption line shape that appear very different, both approximations are shown to yield identical results for the infrared line shape of a harmonic system in the condensed-phase. The equivalence of the FFA and CDA is also demonstrated in the case where the transition dipole of the oscillator fluctuates in response to the environment (i.e., where the Condon approximation has been relaxed). Finally we examine the effects of solute anharmonicity and demonstrate that the CDA and FFA are not equivalent in general, and the magnitude of the deviations increases with anharmonicity. We conclude that the calculation of infrared absorption line shapes via the CDA is a promising alternative to the FFA approach in cases where it may be difficult or undesirable to employ the latter, particularly when the effects of anharmonicity are small.

Nitrile Groups as Vibrational Probes: Calculations of the C≡N Infrared Absorption Line Shape of Acetonitrile in Water and Tetrahydrofuran

The C[TRIPLE BOND]N bond is a powerful probe of protein structure and dynamics because it absorbs in a region of the infrared spectrum apart from the other vibrations that occur naturally in proteins, and because its infrared absorption line shape is sensitive to specific characteristics of the local environment. Since the polarity experienced by the probe can differ dramatically within the protein, infrared spectroscopy of a C[TRIPLE BOND]N site-specifically labeled residue can be used to infer its local environment within the protein. It has been shown experimentally that the spectrum of acetonitrile in water is different in terms of peak position and width compared to acetonitrile in tetrahydrofuran (THF). An optimized quantum mechanics/molecular mechanics method for calculating accurate vibrational frequencies in condensed-phase was parametrized for acetonitrile in water. The transferability of the methodology to a different solvent was tested by computing the infrared line shapes of acetonitrile in both water and THF and comparing to experiment. The infrared absorption line shapes agree well with experiment in each case, and the trends observed experimentally are recovered. The accuracy of the methodology for two solvents of differing polarity indicates that this technique is suitable to study CN probes in proteins.

Sum-frequency generation as a vibrational and electronic probe of the electrochemical interface and thin films

Standard vibrational sum-frequency generation (SFG) spectroscopy is performed to probe the electrochemical cyanoacetylene/Au(1 1 1) interface, enlightening a 20 cm −1/V Stark shift of the free CN vibration mode of this cyanopolyynes-class molecule. SFG data suggest an orientation for the adsorbed molecules with the CN moiety pointing out in a direction perpendicular to the metal surface. A newly developed two-colour SFG (2C-SFG) set-up based on the CLIO free electron laser synchronized with a tuneable visible laser source is used to probe simultaneously the vibrational and electronic fingerprint of a thiophenol/Ag(1 1 1) interface. Structural information on the adsorbed self-assembled monolayer is put in evidence as a function of the immersion time of the silver substrate in the thiophenol solution (24 h or 5 days). With a longer immersion time, the molecular packing is of better quality and more compact, with the carbon rings less tilted with respect to the surface normal. This new experimental set-up combines the advantages to have a higher spectral and temporal resolution with higher power energies at far infrared wavelengths than these presently available from tabletop infrared optical parametric oscillators.

Evidence for Coupling between Nitrile Groups Using DNA Templates: A Promising New Method for Monitoring Structures with Infrared Spectroscopy

Infrared spectroscopy is a common method for monitoring biomolecular structures but suffers from spectral congestion. Non-natural vibrational probes provide a way to regain structural specificity because they provide a unique vibrational signature and can be incorporated into proteins or other biomolecules at specific locations. A popular probe is the nitrile group because its frequency is sensitive to the electrostatics of its environment. In this work, we show that pairs of nitrile groups can be used to directly probe distances and angles in dual labeled molecules. By labeling model DNA oligomers with pairs of nitrile tags, we demonstrate that the vibrational coupling between two nitrile groups is strong enough that Fourier transform infrared (FTIR) spectra can be used to probe relative nitrile distances >4.5 A. Our approach is similar in spirit to monitoring structures with fluorescence resonance energy transfer (FRET) using a pair of fluorescent labels or a pair of spin labels in electron spin resonance spectroscopy. The small sizes of nitrile groups make especially valuable probes of sterically confined regions like the inner cores of large biomolecules where other spectroscopic probes do not fit.

Frequency-frequency correlation functions and apodization in two-dimensional infrared vibrational echo spectroscopy: A new approach

Ultrafast two-dimensional infrared (2D-IR) vibrational echo spectroscopy can probe structural dynamics under thermal equilibrium conditions on time scales ranging from femtoseconds to approximately 100 ps and longer. One of the important uses of 2D-IR spectroscopy is to monitor the dynamical evolution of a molecular system by reporting the time dependent frequency fluctuations of an ensemble of vibrational probes. The vibrational frequency-frequency correlation function (FFCF) is the connection between the experimental observables and the microscopic molecular dynamics and is thus the central object of interest in studying dynamics with 2D-IR vibrational echo spectroscopy. A new observable is presented that greatly simplifies the extraction of the FFCF from experimental data. The observable is the inverse of the center line slope (CLS) of the 2D spectrum. The CLS is the inverse of the slope of the line that connects the maxima of the peaks of a series of cuts through the 2D spectrum that are parallel to the frequency axis associated with the first electric field-matter interaction. The CLS varies from a maximum of 1 to 0 as spectral diffusion proceeds. It is shown analytically to second order in time that the CLS is the T(w) (time between pulses 2 and 3) dependent part of the FFCF. The procedure to extract the FFCF from the CLS is described, and it is shown that the T(w) independent homogeneous contribution to the FFCF can also be recovered to yield the full FFCF. The method is demonstrated by extracting FFCFs from families of calculated 2D-IR spectra and the linear absorption spectra produced from known FFCFs. Sources and magnitudes of errors in the procedure are quantified, and it is shown that in most circumstances, they are negligible. It is also demonstrated that the CLS is essentially unaffected by Fourier filtering methods (apodization), which can significantly increase the efficiency of data acquisition and spectral resolution, when the apodization is applied along the axis used for obtaining the CLS and is symmetrical about tau=0. The CLS is also unchanged by finite pulse durations that broaden 2D spectra.

Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site?

We report the site-specific incorporation of a thiocyanate vibrational probe into the active-site oxyanion hole of ketosteroid isomerase (KSI) to test the effect of hydrophobic steroid binding and solvent exclusion on the local electrostatic environment at this position. While binding of an uncharged ground state steroid analogue shifts the observed −CN vibrational frequency by +0.4 cm-1 relative to unliganded KSI, binding of an intermediate steroid analogue containing localized negative charge results in a +2.8 cm-1 shift. On the basis of a Stark tuning rate of 0.7 cm-1/(MV/cm), this shift indicates a fivefold larger change in the projection of the local electric field along the −CN bond in the presence of the charged ligand. Binding of a single ring phenolate with oxyanion charge localization equivalent to the intermediate steroid analogue but lacking distal hydrocarbon rings results in an identical −CN peak shift. We conclude that solvent exclusion and replacement by hydrophobic steroid rings negligibl...

Energy transport in peptide helices.

We investigate energy transport through an alpha-aminoisobutyric acid-based 3(10)-helix dissolved in chloroform in a combined experimental-theoretical approach. Vibrational energy is locally deposited at the N terminus of the helix by ultrafast internal conversion of a covalently attached, electronically excited, azobenzene moiety. Heat flow through the helix is detected with subpicosecond time resolution by employing vibrational probes as local thermo meters at various distances from the heat source. The experiment is supplemented by detailed nonequilibrium molecular dynamics (MD) simulations of the process, revealing good qualitative agreement with experiment: Both theory and experiment exhibit an almost instantaneous temperature jump of the reporter units next to the heater which is attributed to the direct impact of the isomerizing azobenzene moiety. After this impact event, helix and azobenzene moiety appear to be thermally decoupled. The energy deposited in the helix thermalizes on a subpicosecond timescale and propagates along the helix in a diffusive-like process, accompanied by a significant loss into the solvent. However, in terms of quantitative numbers, theory and experiment differ. In particular, the MD simulation seems to overestimate the heat diffusion constant (2 A(2) ps(-1) from the experiment) by a factor of five.

Vibrational energy in molecules probed with high time and space resolution

This article reviews experimental measurements of vibrational energy in condensed-phase molecules that simultaneously provide time resolution of picoseconds and spatial resolution of ångströms. In these measurements, ultrashort light pulses are used to input vibrational energy and probe dynamical processes. High spatial resolution is obtained using vibrational reporter groups in known locations on the molecules. Three examples are discussed in detail: (1) vibrational energy flow across molecules in a liquid from an OH–group to a CH3–group; (2) vibrational energy flow across a molecular surfactant monolayer that separates an aqueous and a non-polar phase in a suspension of reverse micelles; and (3) vibrational energy input by laser-driven shock waves to a self-assembled monolayer of long-chain alkane molecules. These experiments provide new insights into the movement of mechanical energy over short length and time scales where ordinary concepts of heat conduction no longer apply, where the concepts of quantum mechanical energy transfer reign supreme.

Two-dimensional vibrational optical probes for peptide fast folding investigation.

A simulation study shows that early protein folding events may be investigated by using a recently developed family of nonlinear infrared techniques that combine the high temporal and spatial resolution of multidimensional spectroscopy with the chirality-specific sensitivity of amide vibrations to structure. We demonstrate how the structural sensitivity of cross-peaks in two-dimensional correlation plots of chiral signals of an alpha helix and a beta hairpin may be used to clearly resolve structural and dynamical details undetectable by one-dimensional techniques (e.g. circular dichroism) and identify structures indistinguishable by NMR.

A Genetically Encoded Infrared Probe

An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved that makes it possible to selectively and efficiently incorporate para-cyanophenylalanine (pCNPhe) into proteins in E. coli at sites specified by the amber nonsense codon, TAG. Substitution of pCNPhe for histidine-64 in myoglobin (Mb) affords a sensitive vibrational probe of ligand binding. This methodology provides a useful infrared reporter of protein structure, biomolecular interactions, and conformational changes.

Density Functional Theory (DFT) Calculations of the Infrared Absorption Spectra of Acetaminophen Complexes Formed with Ethanol and Acetone Species

We have investigated the infrared (IR) vibrational spectra of acetaminophen (N(4-hydroxyphenyl) acetamide or paracetamol) complexes formed with ethanol and acetone in relation to the nature of the specific intermolecular interactions involved in the stabilization of the complexes. The structures and binding energies of the complexes have been determined using Hartree-Fock (HF) and DFT-B3PW91 procedures and different Pople's basis sets as well. The main results are presented and discussed by considering the hydroxyl (OH), amino (NH), and carbonyl (CO) chemical groups of acetaminophen interacting with the acetone or ethanol molecules either separately or in conjunction in the complex formation. The frequency shifts and IR intensity variations associated with the internal modes of acetaminophen (namely nu(OH), nu(NH), and nu(CO)) as well as the most pertinent vibrational probes of ethanol (nu(OH)) and acetone (symmetric nu(CO) and nu(CCC) stretching modes) interacting with acetaminophen have been analyzed. The predicted spectral changes have been critically discussed in comparison with IR absorption measurements of acetaminophen dissolved as a solute in ethanol or acetone CO2 expanded solutions. It is argued that the exchange-correlation contribution taken into account in DFT calculations is likely significant in determining the main IR spectral features of acetaminophen complexes formed with acetone or involving hydrogen-bonded as with ethanol.

Ultrafast Dynamics of Shock Compression of Molecular Monolayers

Femtosecond laser-driven approximately 1 GPa shock waves are used to compress monolayers of hydrocarbon chains. Vibrational sum-frequency generation spectroscopy probes the orientation of the terminal methyl groups. With an odd number (15) of carbon atoms, shock compression is an elastic process that causes the methyl groups to tilt. With an even number (18) of carbon atoms, shock compression is viscoelastic, creating single and double gauche defects. When the shock unloads, single gauche defects remain while double defects relax in 30 ps to single-defect states with more upright methyl groups.

Probing molecular dynamics using action, Doppler and photoacoustic spectroscopy

Vibrationally mediated photodissociation involves laser techniques that deposit energy in skeletal motions of molecules, which subsequently interact with photons promoting them to excited electronic states. The molecules adiabatically or non-adiabatically dissociate, releasing atomic photofragments. Action and Doppler spectroscopies, expressing the yield of the ensuing fragments vs. the vibrational excitation and UV probe lasers, respectively, are measured. These spectra together with the simultaneously measured photoacoustic spectra provide a unique means for accessing the dynamics on the ground and excited potential energy surfaces. Experimental studies concerning acetylene and its 1-butyne homologue demonstrate how the mechanism of the chemical transformations and the nature of rovibrationally excited states are highlighted by photolysis of pre-excited molecules.