Vibrational Probe Research Articles

Structural Influences on the Fast Dynamics of Alkylsiloxane Monolayers on SiO2 Surfaces Measured with 2D IR Spectroscopy

There is widespread interest in alkyl chain surface monolayers and their applications. In many applications, alkyl monolayers are functionalized with active headgroups. Here we report the impact of major structural variations on the fast dynamics of alkylsiloxane monolayers. The monolayers were deposited with controlled structures on flat amorphous silica surfaces, and the terminal sites were functionalized with a metal carbonyl headgroup. The CO symmetric stretching mode of the headgroup served as a vibrational probe for detecting the fast structural dynamics of the monolayers using two-dimensional infrared vibrational echo spectroscopy (2D IR) to measure spectral diffusion, which is made quantitative by determining the frequency–frequency correlation function (FFCF) from the time-dependent data. Two methods of functionalizing the surface, independent attachment via a single Si–O bond formed with alkylmonochlorosilane precursors and network attachment via siloxane networks (−Si–O–Si–O−) formed with alkyl...

Ultrafast 2D-IR and simulation investigations of preferential solvation and cosolvent exchange dynamics.

Using a derivative of the vitamin biotin labeled with a transition-metal carbonyl vibrational probe in a series of aqueous N,N-dimethylformamide (DMF) solutions, we observe a striking slowdown in spectral diffusion dynamics with decreased DMF concentration. Equilibrium solvation dynamics, measured with the rapidly acquired spectral diffusion (RASD) technique, a variant of heterodyne-detected photon-echo peak shift experiments, range from 1 ps in neat DMF to ∼3 ps in 0.07 mole fraction DMF/water solution. Molecular dynamics simulations of the biotin-metal carbonyl solute in explicit aqueous DMF solutions show marked preferential solvation by DMF, which becomes more pronounced at lower DMF concentrations. The simulations and the experimental data are consistent with an interpretation where the slowdown in spectral diffusion is due to solvent exchange involving distinct cosolvent species. A simple two-component model reproduces the observed spectral dynamics as well as the DMF concentration dependence, enabling the extraction of the solvent exchange time scale of 8 ps. This time scale corresponds to the diffusive motion of a few Å, consistent with a solvent-exchange mechanism. Unlike most previous studies of solvation dynamics in binary mixtures of polar solvents, our work highlights the ability of vibrational probes to sense solvent exchange as a new, slow component in the spectral diffusion dynamics.

Probing structural features of self-assembled violanthrone-79 using two dimensional infrared spectroscopy.

Two-dimensional infrared (2D IR) spectroscopy was used to characterize the structure of a self-assembled polycyclic aromatic hydrocarbon (PAH), violanthrone-79. A local mode basis was constructed using spectroscopic and computational results of anthrone and monomer violanthrone-79. The vibrational modes in the spectral region 1550-1700 cm(-1), carbonyl stretching and in-plane ring breathing, are used as vibrational probes. The local mode basis and an electrostatic coupling model were applied to three nanoaggregate structures: parallel, antiparallel, and a chiral configuration produced by a 28° rotation from parallel. Angular disorder within each nanoaggregate configuration was also explored. This investigation is a first approach to probe self-assembled PAHs with 2D IR spectroscopy. The experimental and calculated 2D IR spectra align best when the violanthrone-79 molecules are in an anti-parallel configuration within the nanoaggregate.

Vibrational dynamics of azide-derivatized amino acids studied by nonlinear infrared spectroscopy.

Recently, biomolecules which are labeled by azide or thiocyanate groups in solutions and proteins have been studied to examine microscopic environment around a solute by nonlinear infrared (IR) spectroscopy. In this study, we have performed two-dimensional infrared (2D-IR) spectroscopy to investigate the vibrational frequency fluctuations of two different azide-derivatized amino acids, Ala (N3-Ala) and Pro (N3-Pro), and N3(-) in water. From the 2D-IR experiments, it was found that the frequency-frequency time correlation function (FFTCF) of solute can be modeled by a delta function plus an exponential function and constant. FFTCF for each probe molecule has a decay component of about 1 ps, and this result suggests that the stretching mode of the covalently bonded azide group is sensitive to the fluctuations of hydrogen bond network system, as found in previous studies of N3(-) in water. In contrast to FFTCF of N3(-), FFTCF of the azide-derivatized amino acids contains static component. This static component may reflect dynamics of water affected by the solutes or the structural fluctuations of the solute itself. We also performed the IR pump-probe measurements for the probe molecules in water in order to investigate vibrational energy relaxation (VER) and reorientational relaxation. It was revealed that the charge fluctuations in the azide group are significant for the VER of this mode in water, reflecting that the VER rate of N3(-) is faster than those of the azide-derivatized amino acids. While the behaviors of the anisotropy decay of N3-Ala and N3(-) are similar to each other, the anisotropy decay of N3-Pro contains much slower decaying component. By considering the structural difference around the vibrational probe between N3-Ala and N3-Pro, it is suggested that the structural freedom of the probe molecules can affect the reorientational processes.

Low frequency 2D Raman-THz spectroscopy of ionic solution: A simulation study.

The 2D Raman-THz spectrum of the MgCl2 solution was simulated using the molecular dynamics simulation and the stability matrix method and compared with that of the pure water. The 2D Raman-THz signal provides more information on the ion effects on the collective water motion than the conventional 1D signal. The presence of MgCl2 suppresses the cross peak of water between the hydrogen bond bending and the other intermolecular vibrational mode, which clearly illustrates that the water hydrogen bending motion is affected by the confining effect of the ions. Our theoretical work thus demonstrates that the 2D Raman-THz technique can become a valuable nonlinear vibrational probe for the molecular dynamics in the ionic solutions.

Measuring electric fields and noncovalent interactions using the vibrational stark effect.

Over the past decade, we have developed a spectroscopic approach to measure electric fields inside matter with high spatial (<1 Å) and field (<1 MV/cm) resolution. The approach hinges on exploiting a physical phenomenon known as the vibrational Stark effect (VSE), which ultimately provides a direct mapping between observed vibrational frequencies and electric fields. Therefore, the frequency of a vibrational probe encodes information about the local electric field in the vicinity around the probe. The VSE method has enabled us to understand in great detail the underlying physical nature of several important biomolecular phenomena, such as drug-receptor selectivity in tyrosine kinases, catalysis by the enzyme ketosteroid isomerase, and unidirectional electron transfer in the photosynthetic reaction center. Beyond these specific examples, the VSE has provided a conceptual foundation for how to model intermolecular (noncovalent) interactions in a quantitative, consistent, and general manner. The starting point for research in this area is to choose (or design) a vibrational probe to interrogate the particular system of interest. Vibrational probes are sometimes intrinsic to the system in question, but we have also devised ways to build them into the system (extrinsic probes), often with minimal perturbation. With modern instruments, vibrational frequencies can increasingly be recorded with very high spatial, temporal, and frequency resolution, affording electric field maps correspondingly resolved in space, time, and field magnitude. In this Account, we set out to explain the VSE in broad strokes to make its relevance accessible to chemists of all specialties. Our intention is not to provide an encyclopedic review of published work but rather to motivate the underlying framework of the methodology and to describe how we make and interpret the measurements. Using certain vibrational probes, benchmarked against computer models, it is possible to use the VSE to measure absolute electric fields in arbitrary environments. The VSE approach provides an organizing framework for thinking generally about intermolecular interactions in a quantitative way and may serve as a useful conceptual tool for molecular design.

Kinetics of exchange between zero-, one-, and two-hydrogen-bonded states of methyl and ethyl acetate in methanol.

It has recently been shown that the ester carbonyl stretching vibration can be used as a sensitive probe of local electrostatic field in molecular systems. To further characterize this vibrational probe and extend its potential applications, we studied the kinetics of chemical exchange between differently hydrogen-bonded (H-bonded) ester carbonyl groups of methyl acetate (MA) and ethyl acetate (EA) in methanol. We found that, while both MA and EA can form zero, one, or two H-bonds with the solvent, the population of the 2hb state in MA is significantly smaller than that in EA. Using a combination of linear and nonlinear infrared measurements and numerical simulations, we further determined the rate constants for the exchange between these differently H-bonded states. We found that for MA the chemical exchange reaction between the two dominant states (i.e., 0hb and 1hb states) has a relaxation rate constant of 0.14 ps(-1), whereas for EA the three-state chemical exchange reaction occurs in a predominantly sequential manner with the following relaxation rate constants: 0.11 ps(-1) for exchange between 0hb and 1hb states and 0.12 ps(-1) for exchange between 1hb and 2hb states.

Site-specific infrared probes of proteins.

Infrared spectroscopy has played an instrumental role in the study of a wide variety of biological questions. However, in many cases, it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and environmental information in a site-specific manner. To overcome this limitation, investigators have dedicated many recent efforts to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural or environmental properties. In this review, we highlight recent advancements in this rapidly growing research area.

Perspectives on electrostatics and conformational motions in enzyme catalysis.

ConspectusEnzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR).We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH.Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors.

Site-Specific Conformational Diversity in Calmodulin using Cyanylated Cysteine Vibrational Probes along its Binding Interfaces

Vibrational spectroscopy was used to probe the modular binding interfaces of calmodulin. Two hydrophobic amino acid patches allow calmodulin great flexibility in binding to target partners, and these patches contain key methionine residues. Site-specific mutagenesis was used to generate numerous single cysteine variants of calmodulin, including three mutations at key methionine residues, which were expressed, purified and cyanylated at cysteine to generate a vibrational probe group at each site. Infrared spectroscopy was used to assess the environment surrounding each probe in apo- and calcium saturated conditions, as well as complexed with the binding peptide from skeletal muscle myosin light chain kinase. Circular dichroism (CD) experiments were conducted to ensure the SCN vibrational probe did not perturb calmodulin's secondary structure, and isothermal calorimetry was performed for each labeled variant with the skMLCK peptide to ensure the probe did not disrupt complex formation. Surprisingly, the methionine to cyanylated cysteine mutation at key methionine residues did not lead to a large perturbation in binding thermodynamics. This suggests that the cyanylated cysteine probe group is relatively innocent even when placed directly in the binding interface and can report directly on structural dynamics along the binding interface between calmodulin and its many targets.

Allostery in PDZ3: Using Unnatural Amino Acids as Site-Specific Reporters in IR Spectroscopy to Probe Allosteric Pathways

The third PDZ domain of the multidomain complex PSD-95 is a well studied but controversially discussed model for dynamic allostery without major conformational changes. After the first predictions of a coupled amino acid network by Ranganathan and Lockless1 followed by Ota and Agard2, that is potentially involved in intradomain signaling and allostery, several computational and experimental attempts were made to gain insight into the presence or absence of a wire-like pathway for information transfer in PDZ33-5. A recent development in vibrational spectroscopy enables us to revisit this long-standing question with a new experimental approach. Using the unnatural amino acid Azidohomoalanine (Aha) as vibrational probe at different positions in PDZ3, we tested the influence of ligand binding on the local microenvironment of the probe. As the azide absorption band of Aha is sensitive to even subtle changes of its environment6-7, this approach allows us to monitor conformational fluctuations invisible to other methods. The amino acid residues tested, that are part of the predicted coupled network indeed show a change of the local microenvironment not reported experimentally before. In contrast no differences were found for residues which are not part of the predicted pathway but at similar distances from the binding pocket. This is an experimental evidence for long range communication and allostery in PDZ3.1Lockless, S. W. et al., Science 1999, 286, 294-2992Ota, N. et al., J. Mol. Biol. 2005, 351, 345-3543Chi, C. N. et al., Biochemistry 2012, 51, 8971-89794Chi, C. N. et al., PNAS, 105, 12, 4679-46845Petit, C. M. et al., PNAS 2009, 106, 43, 18249-182546Taskent-Sezgin, H. et al., Angew. Chem. Int. Ed. 2010, 49, 7473-74757Choi, J.-H. et al., JPCL 2011, 2, 2158-2162

Assessing Binding Perturbation due to Artificial Vibrational Probe Groups in the Nucleoprotein-Phosphoprotein Complex of the Nipah Virus

The binding interaction between the intrinsically disordered nucleoprotein tail and the phosphoprotein of the Nipah Virus (NiV) involves both disorder-to-order transition and fuzzy binding. To examine the dynamic structure and the conformational distribution of this interaction, a site-specific thiocyanate (SCN) vibrational probe was incorporated at many sites on the binding region of the NiV NTAIL. Since this binding is likely driven by hydrophobic forces, replacing a non-polar amino acid side chain with a polar probe could perturb binding. Isothermal titration calorimetry (ITC) experiments were designed to determine the extent of disruption to binding thermodynamics. The ITC results were then used to inform the interpretation of the vibrational spectroscopy data and measure the importance of single amino acids in maintaining this “fuzzy” binding interface.

Developing a Protocol for Ensemble and Vibrational Probe-Containing Molecular Dynamics Simulations of the Nipah Ntail-XD Complex

Intrinsically disordered proteins (IDPs) prove difficult to characterize using typical protein secondary structure indicators, such as CD and NMR spectroscopy, because of their dynamic nature. Molecular Dynamics (MD) simulations can be adapted for use in highly dynamic systems such as IDPs through enhanced sampling techniques. This project involves the characterization of the “fuzzy” Nipah Virus nucleo- and phosphoprotein bound complex by using MD simulations and vibrational probes. In order to run MD simulations on IDPs, a viable structural ensemble must be generated; this can be done by varying the simulation temperature to overcome energy boundaries and exchanging high-energy structures with those generated at low temperature (“replica exchange”). Once an ensemble of structures is generated, shorter simulations can be run in which a site-specific thiocyanate probe is incorporated into each structure. Eventually, an infrared (IR) lineshape can be simulated from the conformational ensemble for each label site and directly compared to experimental data. Progress towards each step of this multi-step simulation protocol will be discussed, as well as the prospects for a hybrid spectroscopic-theoretical determination of the conformational distribution of “fuzzy” bound complexes.

Studying the Membrane-Bound Conformation of Alpha-Synuclein using a Model Transmembrane Peptide System in a Lipid Bilayer

Alpha-synuclein is intrinsically disordered in solution but adopts a partially alpha-helical structure when membrane bound; we are currently investigating this structure using a cyanylated cysteine vibrational probe at various sites on the αS membrane binding domain. In order to quantitatively understand the depth dependence of the SCN probe in a lipid bilayer, four variants of a poly-L transmembrane helical peptide were designed and synthesized with cysteine residues at different depths in a membrane bilayer. A nonaqueous cyanylation and purification protocol was developed to introduce the probe group on these very hydrophobic model peptides. Vibrational spectroscopy was carried out on the four variants in a model lipid system using both solution/vesicle and oriented, immobilized samples. These data will help to elucidate the membrane interactions of both αS and other membrane-bound proteins.

Applications of two-dimensional infrared spectroscopy.

Two-dimensional infrared (2D IR) spectroscopy has recently emerged as a powerful tool with applications in many areas of scientific research. The inherent high time resolution coupled with bond-specific spatial resolution of IR spectroscopy enable direct characterization of rapidly interconverting species and fast processes, even in complex systems found in chemistry and biology. In this minireview, we briefly outline the fundamental principles and experimental procedures of 2D IR spectroscopy. Using illustrative example studies, we explain the important features of 2D IR spectra and their capability to elucidate molecular structure and dynamics. Primarily, this minireview aims to convey the scope and potential of 2D IR spectroscopy by highlighting select examples of recent applications including the use of innate or introduced vibrational probes for the study of nucleic acids, peptides/proteins, and materials.

New divergent dynamics in the isotropic to nematic phase transition of liquid crystals measured with 2D IR vibrational echo spectroscopy.

The isotropic phase of nematogenic liquid crystals has nanometer length scale domains with pseudonematic ordering. As the isotropic to nematic phase transition temperature (TNI) is approached from above, the orientational correlation length, ξ, of the pseudonematic domains grows as (T - T(*))(-1/2), where T(*) is 0.5-1 K below TNI. The orientational relaxation, which is a collective property of the pseudonematic domains, was measured with optical heterodyne detected-optical Kerr effect (OHD-OKE). The orientational relaxation obeys Landau-de Gennes theory, as has been shown previously. To examine the environmental evolution experienced by molecules in the pseudonematic domains, two-dimensional infrared (2D IR) vibrational echo experiments on the CN stretching mode of the non-perturbative vibrational probes 4-pentyl-4(')-selenocyanobiphenyl (5SeCB) and 4-pentyl-4(')-thiocyanobiphenyl (5SCB) in the nematogen 4-cyano-4(')-pentylbiphenyl (5CB) were performed. The 2D IR experiments measure spectral diffusion, which is caused by structural fluctuations that couple to the CN vibrational frequency. Temperature dependent studies were performed just above TNI, where the correlation length of pseudonematic domains is large and changing rapidly with temperature. These studies were compared to 2D IR experiments on 4-pentylbiphenyl (5B), a non-mesogenic liquid that is very similar in structure to 5CB. The time constants of spectral diffusion in 5CB and 5B are practically identical at temperatures ≥5 K above TNI. As the temperature is lowered, spectral diffusion in 5B slows gradually. However, the time constants for spectral diffusion in 5CB slow dramatically and diverge as T(*) is approached. This divergence has temperature dependence proportional to (T - T(*))(-1/2), precisely the same as seen for the correlation length of pseudonematic domains, but different from the observed orientational relaxation times, which are given by the Landau-de Gennes theory. The data and previous results show that spectral diffusion in 5CB has no contributions from orientational relaxation, and the structural dynamics responsible for the spectral diffusion are likely a result of density fluctuations. The results suggest that the correlation length of the density fluctuations is diverging with the same temperature dependence as the pseudonematic domain correlation length, ξ. The isotropic-nematic phase transition in liquid crystals is described in the context of the slowing of orientational relaxation associated with divergent growth of the orientational correlation length. The results presented here show that there is another divergent dynamical process, likely associated with density fluctuations.

Characterizing Solvent Dynamics in Nanoscopic Silica Sol–Gel Glass Pores by 2D-IR Spectroscopy of an Intrinsic Vibrational Probe

Porous sol–gel matrices were synthesized with IR-active Si–H vibrational chromophores integrated into the silica network. The Si–H vibrational mode was found to be highly accessible to solvents within the nanoscopic pores. Vibrational solvatochromism of the silane vibration was controlled largely by interactions between infiltrating solvents and the oxygen and hydroxide sites in the silica. Exchanging solvents in the silica matrices produced reversible solvatochromic shifts in some cases but led to irreversible shifts when strongly interacting solvents were tested, suggesting that a layer of solvent was not exchangeable. 2D-IR spectroscopy was used to monitor spectral diffusion and extract the homogeneous line widths of the Si–H mode for a range of infiltrating solvents as well as solvent-free aerogel samples. It was demonstrated that the silane vibration is sensitive to the nature of the infiltrating solvent, making these vibrationally active sol–gel films a general platform for solvent dynamics in nanoscopic confined volumes.

Vibrational coherence probes the mechanism of ultrafast electron transfer in polymer-fullerene blends.

The conversion of photoexcitations into charge carriers in organic solar cells is facilitated by the dissociation of excitons at the donor/acceptor interface. The ultrafast timescale of charge separation demands sophisticated theoretical models and raises questions about the role of coherence in the charge-transfer mechanism. Here, we apply two-dimensional electronic spectroscopy to study the electron transfer process in poly(3-hexylthiophene)/PCBM (P3HT/PCBM) blends. We report dynamics maps showing the pathways of charge transfer that clearly expose the significance of hot electron transfer. During this ultrafast electron transfer, vibrational coherence is directly transferred from the P3HT exciton to the P3HT hole polaron in the crystalline domain. This result reveals that the exciton converts to a hole with a similar spatial extent on a timescale far exceeding other photophysical dynamics including vibrational relaxation.

Solvent induced conformational fluctuation of alanine dipeptide studied by using vibrational probes

The solvation effect on the three dimensional structure and the vibrational feature of alanine dipeptide (ALAD) was evaluated by applying the implicit solvents from polarizable continuum solvent model (PCM) through ab initio calculations, by using molecular dynamic (MD) simulations with explicit solvents, and by combining these two approaches. The implicit solvent induced potential energy fluctuations of ALAD in CHCl3, DMSO and H2O are revealed by means of ab initio calculations, and a global view of conformational and solvation environmental dependence of amide I frequencies is achieved. The results from MD simulations with explicit solvents show that ALAD trends to form PPII, αL, αR, and C5 in water, PPII and C5 in DMSO, and C5 in CHCl3, ordered by population, and the demonstration of the solvated structure, the solute–solvent interaction and hydrogen bonding is therefore enhanced. Representative ALAD–solvent clusters were sampled from MD trajectories and undergone ab initio calculations. The explicit solvents reveal the hydrogen bonding between ALAD and solvents, and the correlation between amide I frequencies and the CO bond length is built. The implicit solvents applied to the ALAD–solvent clusters further compensate the solvation effect from the bulk, and thus enlarge the degree of structural distortion and the amide I frequency red shift. The combination of explicit solvent in the first hydration shell and implicit solvent in the bulk is helpful for our understanding about the conformational fluctuation of solvated polypeptides through vibrational probes.

Photoisomerization and structural dynamics of two nitrosylruthenium complexes: a joint study by NMR and nonlinear IR spectroscopies.

In this work, the photoisomerization and structural dynamics of two isomeric nitrosylruthenium(ii) complexes [Ru(OAc)(2cqn)2NO] (H2cqn = 2-chloro-8-quinolinol) in CDCl3 and DMSO are examined using NMR and IR spectroscopic methods. The two N atoms in the 2cqn ligand are in trans position in the synthesized cis-1 isomer, while they are in cis position in the cis-2 isomer. Kinetics monitored by NMR spectroscopy shows that the rate constant of photoisomerization from cis-2 to cis-1 isomer depends on the wavelength of irradiation and solvent polarity; it proceeds faster on irradiating near the absorption peak in the UV-Vis region, and also in more polar solvents (DMSO). Density functional theory computation indicates that the peculiarity of [Ru(ii)-NO(+)] group affects the structure and reactivity of the nitrosylruthenium complexes. Using the nitrosyl stretching (νNO) to be vibrational probe, the structural dynamics and structural distributions of the cis-1 and cis-2 isomers are examined by steady-state linear infrared and ultrafast two-dimensional infrared (2D IR) spectroscopies. The structural and photochemical aspects of the observed spectroscopic parameters are discussed in terms of solute-solvent interactions for the two nitrosylruthenium complexes.