2D IR Spectra Research Articles

Synthesis of 5-Cyano-Tryptophan as a Two-Dimensional Infrared Spectroscopic Reporter of Structure.

A concise synthesis of protected 5-cyano-l-tryptophan (Trp5CN ) has been developed for 2D IR spectroscopic investigations within either peptides or proteins. To assess the potential of differently substituted cyano-tryptophans, several model cyano-indole systems were characterized using IR spectroscopy. Upon assessment of their spectroscopic properties, Trp5CN was integrated into a model peptide sequence, Trp5CN -Gly-Phe4CN , to elucidate its structure. This peptide demonstrates the capability of this probe to capture structural information by 2D IR spectroscopy. The 2D IR spectrum of the peptide in water was simulated to reveal a unique spectral signature resulting from the presence of dipolar coupling. The coupling strength between cyano labels was determined to be 1.4 cm-1 by matching the slopes along the max contour for the simulated and experimental spectrum. Using transition dipole coupling, a distance between the two probes of 13 Å was calculated.

Two-dimensional infrared study of the C[sbnd]D and C[dbnd]O stretching vibrations in strongly hydrogen-bonded complexes

We report a study of the CD and CO stretching modes of formyl deuterated formic acid (DCOOH) hydrogen bonded to nitrogenous bases. These modes are spectators to the hydrogen bond itself but are sensitive to the strength of that interaction. Using 2D IR spectroscopy, we show that in strongly hydrogen bonded complexes the CD stretching vibration becomes anharmonically coupled to low-order combination states leading to strong state mixing resulting in Fermi resonance transitions in the 2D IR spectra. In contrast, the CO vibrations show multiple peaks as a result of conformational heterogeneity associated with internal rotation about the hydrogen bond. For both modes, the population relaxation rates tend to be quite short, ∼1 ps or less. Having characterized the vibrational spectroscopy of these spectator vibrations, we conclude that they are quite sensitive to details of the hydrogen bonding interactions though their utility is limited by spectral complexity and short population lifetimes.

Surface-Enhanced Dual-Frequency Two-Dimensional Vibrational Spectroscopy of Thin Layers at an Interface

The development of spectroscopic approaches to study molecules at interfaces is important as the molecular properties often differ from those in the bulk. Implementation of surface-enhanced two-dimensional infrared (SE 2DIR) spectroscopy using lithographically fabricated plasmonic nanoarrays is demonstrated for nanometer thick films. The sample, 4-azidobutyrate-N-hydroxysuccinimide ester (azNHS), dispersed in polystyrene was deposited onto the nanoarray. Raw enhancements in the SE 2DIR spectra exceeding 5 × 104 and 1.3 × 103 fold were achieved for the CO and NN peaks, respectively. The field enhancement provided by the nanoarray was sufficient to record cross-peaks in 1 nm thick samples under dilute conditions for azNHS (∼0.1 M). Note that the cross-peaks were recorded for vibrational modes frequency separated by ∼350 cm–1 with the enhancement factor of 4.1 × 104. The effective electric field enhancement factors, measured for NN and CO modes via linear and two nonlinear IR techniques, have similar sample-...

Identification of cattail pollen, pine pollen and bee pollen by Fourier transform infrared spectroscopy and two-dimensional correlation infrared spectroscopy

Cattail pollen (CP) and pine pollen (PP) are traditional Chinese medicine, which are used for bleeding blood and immunity enhancement activity respectively. Due to the economic benefits, they are occasionally adulterated with bee pollen. In this study, CP, PP and 6 kinds of bee pollen (dandelion pollen, sunflower pollen, lotus pollen, rhus pollen, buckwheat pollen, corn pollen) were collected and observed by the scanning electron microscope. And then, Fourier transform infrared (FT-IR) spectroscopy and two-dimensional correlation infrared (2D-IR) spectroscopy were used to identify and analyze these 8 kinds of pollens. It is found that PP and CP could be identified from bee pollen through the position, intensity and shape of saccharide and protein characteristic peaks in the FT-IR spectra. The FT-IR spectra of PP, CP and bee pollen are almost similar. 2D-IR spectra exhibited significant differences in automatic peak positions, especially in the range of 760–1400 cm−1. CP and PP have three consistently strong automatic peaks around 1150, 1204, and 1233 cm−1. The sequences of the absorption intensities of them all are 1204 cm−1 > 1233 cm−1 > 1150 cm−1. While CP has no obvious strong automatic peaks compared with PP in the range of 830–1140 cm−1. In conclusion, FT-IR and 2D-IR technology are the effective method for distinguish CP and PP from these 6 kinds of bee pollen.

Two dimensional infrared spectroscopic investigations of protein and enzyme dynamics

Ultra fast two dimensional infrared (2D IR) spectroscopy is used to measure dynamics and interactions in complex materials in the ground electronic state under thermal equilibrium conditions. Two observables, ultra fast chemical exchange and spectral diffusion, will be discussed. In a 2D IR experiment, three variably time delayed IR excitation pulses, tuned to the vibrational transition of interest, give rise to a fourth vibrational echo pulse, the signal in the experiment. The first two pulses in the sequence label the vibrations with their initial frequencies. After a waiting period, Tw, the third pulse induces the vibrational echo, which reads out the final vibrational frequencies. Structural changes of the system cause the vibrational frequencies to change. Therefore, measuring the time evolution of the vibrational frequencies reports on the time evolution of the structure. Folded proteins can exist in multiple conformational substates. Each substate reflects a local minimum on the free‐energy landscape with a distinct structure. Using 2D‐IR chemical exchange spectroscopy, conformational switching between two well defined substates of two myoglobin mutants were observed on the ~50 ps time scale. The conformational dynamics were measured through the growth of off‐diagonal peaks in the 2D IR spectra of CO bound to the heme. The conformational switching likely involves motion of the distal histidine/E helix that changes the position of the histidine's imidazole side group. These results demonstrate that interconversion between protein conformational substates can occur on very fast time scales. Detailed MD simulations are able to capture some of the observed behavior, but not quantitatively. Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. 2D IR spectroscopy was used to measure the dynamics of cyt P450cam from Pseudomonas putida using CO bound at the active site as the vibrational probe. The experiments measure spectral diffusion, the time evolution of the CO vibrational frequencies within the in homogeneously broadened vibrational absorption line. The time evolution of the frequencies is caused by the time evolution of the enzyme structure. The enzyme bound to both its natural substrate, camphor, and a series of related substrates were investigated to explicate the role of dynamics in molecular recognition in cyt P450cam and to delineate how the motions may contribute to hydroxylation specificity. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450cam toward different substrates.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation on intrinsic quality of Wolfiporia cocos influenced by cultivation methods by a multi-step IR macro-fingerprinting technique

Wolfiporia cocos is a well-known traditional East-Asian medicinal fungus. In this study, four Wolfiporia cocos samples (FC-DM, RC-DM, FC-YN and SC-YN) of three cultivation methods harvest from Dabie Mountain area and Yunnan province were discriminated and identified by a multi-step IR macro-fingerprinting technique. The correlation coefficient of the three cultivation methods of Wolfiporia cocos are higher than 0.98 in the range of 450–2600 cm−1. According to retrieving the spectrum in Sadtler database, the main compositions of Wolfiporia cocos are laminarin polysaccharide. While Correlation coefficients of synchronous 2D-IR spectra of FC-DM and other Wolfiporia cocos samples were furtherly calculated, acquiring a quantitative discrimination result. The thermal sensitivity of polysaccharides of SC-YN is different from other samples in the range of 850–1370 cm−1. The difference in thermal sensitivity of CH (1402 and 1474 cm−1) and amino acid (1666 or 1651 cm−1) could also be considered as the key factors to discriminate them. The result proved that the 2DCOS-IR could discriminate Wolfiporia cocos of different cultivation methods.

Coordination to lanthanide ions distorts binding site conformation in calmodulin

The Ca2+-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca2+ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca2+ and several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tb3+ is commonly used as a luminescent Ca2+ analog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Ca2+ and the lanthanides in CaM. Although functional assays indicate that Tb3+ fully activates many Ca2+-dependent proteins, our FTIR spectra indicate that Tb3+, La3+, and Lu3+ disrupt the bidentate coordination geometry characteristic of the CaM binding sites' strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca2+-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca2+-CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM's binding sites.

Ultrafast Two-Dimensional Infrared Spectroscopy of a Quasifree Rotor: J Scrambling and Perfectly Anticorrelated Cross Peaks.

Ultrafast two-dimensional infrared (2DIR) spectra of the N_{2}O ν_{3} mode in moderately dense SF_{6} gas exhibit complex line shapes with diagonal and antidiagonal features in contrast to condensed phase vibrational 2DIR spectroscopy. Observed spectra for this quasifree rotor system are well captured by a model that includes all 36 possible rovibrational pathways and treats P (ΔJ=-1) and R (ΔJ=+1) branch resonances as distinct Kubo line shape features. Transition frequency correlation decay is due to J scrambling within one to two gas collisions at each density. Studies of supercritical solvation and relaxation at high pressure and temperature are enabled by this methodology.

Vibrational heavy atom effect controls relaxation and spectral diffusion in triphenyl hydride complexes

A series of three triphenyl hydride compounds was investigated in a range of solvents by Fourier transform infrared (FTIR), IR pump-probe, and two-dimensional IR (2D-IR) spectroscopies. The mass of the central atom of the three compounds was varied systematically down the group 14 elements of silicon, germanium, and tin. FTIR studies provided evidence that the solvent perturbs the three hydride modes in a similar manner, as indicated by identical solvatochromic and broadening trends. IR pump-probe measurements showed that the vibrational lifetime of the hydride stretch increased when the mass of the central atom increased. Density functional theory calculations asserted that the vibrational modes of the three compounds are nearly identical except for the hydride stretching and wagging modes. Interestingly, frequency-frequency correlation functions obtained from 2D-IR spectra indicated that an increasingly large central atom produces small, but measurable changes in the dynamics of the solvation shell surrounding each compound.

Rapid Screening of DNA-Ligand Complexes via 2D-IR Spectroscopy and ANOVA-PCA.

Two-dimensional infrared spectroscopy (2D-IR) is well established as a specialized, high-end technique for measuring structural and solvation dynamics of biological molecules. Recent technological developments now make it possible to acquire time-resolved 2D-IR spectra within seconds, and this opens up the possibility of screening-type applications comparing spectra spanning multiple samples. However, such applications bring new challenges associated with finding accurate, efficient methodologies to analyze large data sets in a timely, informative manner. Here, we demonstrate such an application by screening 2016 2D-IR spectra of 12 double-stranded DNA oligonucleotides obtained in the presence and absence of binding therapeutic molecule Hoechst 33258. By applying analysis of variance combined with principal component analysis (ANOVA-PCA) to 2D-IR data for the first time, we demonstrate the ability to efficiently retrieve the base composition of a DNA sequence and discriminate ligand-DNA complexes from unbound sequences. We further show accurate differentiation of the induced-fit and rigid-body binding modes that is key to identifying optimal binding interactions of Hoechst 33258, while ANOVA-PCA results across the full sequence range correlate directly with thermodynamic indicators of ligand-binding strength that require significantly longer data acquisition times to obtain.

Cataracts Contain Amyloid β-Sheets: A 2D IR Study of Human Cataract Tissue

Age-related cataract disease affects the sight of more than 50% of Americans over the age of 80. Cataracts are caused by the aggregation of crystallin proteins in the eye lens, leading to blurred vision, but there is not a clear understanding of the molecular structure changes that lead to the disease. In vitro, crystallin proteins form both amorphous aggregates and amyloid fiber structures, depending on the solution conditions; however, there is little evidence that the aggregates in cataractous lens tissue contain amyloid deposits. This talk will present two-dimensional infrared (2D IR) spectra of natural human cataract lens tissue collected from a deceased individual. 2D IR spectra distinguish between the native β-sheet structures of crystallin lens proteins and their amyloid fiber aggregates through a distinct diagonal peak as well as an off-diagonal cross peak. Control experiments are presented on in vitro crystallin proteins, pig lens tissue, and human non-cataract lens tissue for comparison with diagnosed cataract lens tissue spectra. The 2D IR signatures of amyloid β-sheets are clearly observed in cataract tissue. We also show that exposure to UV radiation, a significant risk factor for age-related cataract disease, produces signatures of amyloid formation in healthy eye lens tissue. These results establish cataracts as an amyloid disease, and have implications for the recent development of small molecules that appear to dissolve cataracts in animal models.

Evidence of Molecular Heterogeneities in Amide-Based Deep Eutectic Solvents.

The liquid structure of five different amide-based deep eutectic solvents (DESs) as a function of the chemical structure of the hydrogen bond acceptor (HBA) was investigated by linear Fourier transform infrared (FTIR) and two-dimensional infrared (2DIR) spectroscopies. Linear FTIR spectroscopy shows that the amide band of the DESs is not significantly affected by the chemical structure and symmetry of the HBA cation. However, its excitonic nature does not allow us to draw further conclusions. Analysis of the 13C amide line shapes derived from the 2DIR spectra reveals that the different DESs do not show appreciable differences in the level of disorganization. The vibrational dynamics, derived from the photon echo experiments on the 13C amide, shows that there is a fast component with a time scale of ∼1 ps irrespective of the HBA. The ultrafast dynamics is assigned to hydrogen bond making and breaking between amides. In addition, a slow dynamical component is observed in the time evolution of the photon echo signal. This contribution appears to be correlated with the asymmetry and polarity of the moieties of the HBA. The overall dynamics is rationalized in terms of a microscopic heterogeneous structure of the DESs, where the heterogeneities create domains that slow the hydrogen bond making and breaking. Molecular dynamics simulations provide additional support for our modeling of the data. In addition, the presence of nanoscopic heterogeneities is consistent with the observation of an endortherm at 23 °C in the differential scanning calorimetry thermogram, which evidenced a phase transition at 23 °C, even though the tested DESs have a melting temperature below -40 °C.

Vibrational Dynamics and Couplings of the Hydrated RNA Backbone: A Two-Dimensional Infrared Study.

The equilibrium structure of the RNA sugar-phosphate backbone and its hydration shell is distinctly different from hydrated DNA. Applying femtosecond two-dimensional infrared (2D-IR) spectroscopy in a range from 950 to 1300 cm-1, we elucidate the character, dynamics, and couplings of backbone modes of a double-stranded RNA A-helix geometry in its aqueous environment. The 2D-IR spectra display a greater number of backbone modes than for DNA, with distinctly different lineshapes of diagonal peaks. Phosphate-ribose interactions and local hydration structures are reflected in the complex coupling pattern of RNA modes. Interactions with the fluctuating water shell give rise to spectral diffusion on a 300 fs time scale, leading to a quasi-homogeneous line shape of the symmetric (PO2)- stretching mode of the strongly hydrated phosphate groups. The RNA results are benchmarked by 2D-IR spectra of DNA oligomers in water and analyzed by molecular dynamics and quantum mechanical molecular mechanics simulations.

Spectroscopic Signature for Stable β-Amyloid Fibrils versus β-Sheet-Rich Oligomers.

We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the β-amyloid (Aβ1-40 and Aβ1-42) protein associated with Alzheimer's disease. Two-dimensional IR spectra resolve a transition at 1610 cm-1 in Aβ fibrils that does not appear in other Aβ aggregates, even those with predominantly β-sheet-structure-like oligomers. This transition is not resolved in linear IR spectroscopy because it lies under the broad band centered at 1625 cm-1, which is the traditional infrared signature for amyloid fibrils. The feature is prominent in 2D IR spectra because 2D lineshapes are narrower and scale nonlinearly with transition dipole strengths. Transmission electron microscopy measurements demonstrate that the 1610 cm-1 band is a positive identification of amyloid fibrils. Sodium dodecyl sulfate micelles that solubilize and disaggregate preaggregated Aβ samples deplete the 1625 cm-1 band but do not affect the 1610 cm-1 band, demonstrating that the 1610 cm-1 band is due to very stable fibrils. We demonstrate that the 1610 cm-1 transition arises from amide I modes by mutating out the only side-chain residue that could give rise to this transition, and we explore the potential structural origins of the transition by simulating 2D IR spectra based on Aβ crystal structures. It was not previously possible to distinguish stable Aβ fibrils from the less stable β-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimer's research on Aβ aggregation, fibril formation, and toxicity.

Revealing the Solvation Structure and Dynamics of Carbonate Electrolytes in Lithium-Ion Batteries by Two-Dimensional Infrared Spectrum Modeling.

Carbonate electrolytes in lithium-ion batteries play a crucial role in conducting lithium ions between two electrodes. Mixed solvent electrolytes consisting of linear and cyclic carbonates are commonly used in commercial lithium-ion batteries. To understand how the linear and cyclic carbonates introduce different solvation structures and dynamics, we performed molecular dynamics simulations of two representative electrolyte systems containing either linear or cyclic carbonate solvents. We then modeled their two-dimensional infrared (2DIR) spectra of the carbonyl stretching mode of these carbonate molecules. We found that the chemical exchange process involving formation and dissociation of lithium-ion/carbonate complexes is responsible for the growth of 2DIR cross peaks with increasing waiting time. In addition, we also found that cyclic carbonates introduce faster dynamics of dissociation and formation of lithium-ion/carbonate complexes than linear carbonates. These findings provide new insights into understanding the lithium-ion mobility and its interplay with solvation structure and ultrafast dynamics in carbonate electrolytes used in lithium-ion batteries.

Ultrafast to Ultraslow Dynamics of a Langmuir Monolayer at the Air/Water Interface Observed with Reflection Enhanced 2D IR Spectroscopy.

Monolayers play important roles in naturally occurring phenomena and technological processes. Monolayers at the air/water interface have received considerable attention, yet it has proven difficult to measure monolayer and interfacial molecular dynamics. Here we employ a new technique, reflection enhanced two-dimensional infrared (2D IR) spectroscopy, on a carbonyl stretching mode of tricarbonylchloro-9-octadecylamino-4,5-diazafluorenerhenium(I) (TReF18) monolayers at two surface densities. Comparison to experiments on a water-soluble version of the metal carbonyl headgroup shows that water hydrogen bond rearrangement dynamics slow from 1.5 ps in bulk water to 3.1 ps for interfacial water. Longer time scale fluctuations were also observed and attributed to fluctuations of the number of hydrogen bonds formed between water and the three carbonyls of TReF18. At the higher surface density, two types of TReF18 minor structures are observed in addition to the main structure. The reflection method can take usable 2D IR spectra on the monolayer within 8 s, enabling us to track the fluctuating minor structures' appearance and disappearance on a tens of seconds time scale. 2D IR chemical exchange spectroscopy further shows these structures interconvert in 30 ps. Finally, 2D spectral line shape evolution reveals that it takes the monolayers hours to reach macroscopic structural equilibrium.

Evaluation on the concentration change of paeoniflorin and glycyrrhizic acid in different formulations of Shaoyao-Gancao-Tang by the tri-level infrared macro-fingerprint spectroscopy and the whole analysis method

As a kind of common prescriptions, Shaoyao-Gancao-Tang (SGT) contains two Chinese herbs with four different proportions which have different clinical efficacy because of their various components. In order to investigate the herb-herb interaction mechanisms, we used the method of tri-level infrared macro-fingerprint spectroscopy to evaluate the concentration change of active components of four SGTs in this research. Fourier transform infrared spectroscopy (FT-IR) and Second derivative infrared spectroscopy (SD-IR) can recognize the multiple prescriptions directly and simultaneously. 2D-IR spectra enhance the spectral resolution and obtain much new information for discriminating the similar complicated samples of SGT. Furthermore, the whole analysis method from the analysis of the main components to the specific components and the relative content of the components may evaluate the quality of TCM better. Then we concluded that paeoniflorin and glycyrrhizic acid were the highest proportion in active ingredients in SGT-12:1 and the lowest one in SGT-12:12, which matched the HPLC-DAD results. It is demonstrated that the method composed by the tri-level infrared macro-fingerprint spectroscopy and the whole analysis can be applicable for effective, visual and accurate analysis and identification of very complicated and similar mixture systems of traditional Chinese medicine.

C-Terminal Truncated α-Synuclein Fibrils Contain Strongly Twisted β-Sheets.

C-terminal truncations of monomeric wild-type alpha-synuclein (henceforth WT-αS) have been shown to enhance the formation of amyloid aggregates both in vivo and in vitro and have been associated with accelerated progression of Parkinson’s disease (PD). The correlation with PD may not solely be a result of faster aggregation, but also of which fibril polymorphs are preferentially formed when the C-terminal residues are deleted. Considering that different polymorphs are known to result in distinct pathologies, it is important to understand how these truncations affect the organization of αS into fibrils. Here we present high-resolution microscopy and advanced vibrational spectroscopy studies that indicate that the C-terminal truncation variant of αS, lacking residues 109–140 (henceforth referred to as 1–108-αS), forms amyloid fibrils with a distinct structure and morphology. The 1–108-αS fibrils have a unique negative circular dichroism band at ∼230 nm, a feature that differs from the canonical ∼218 nm band usually observed for amyloid fibrils. We show evidence that 1–108-αS fibrils consist of strongly twisted β-sheets with an increased inter-β-sheet distance and a higher solvent exposure than WT-αS fibrils, which is also indicated by the pronounced differences in the 1D-IR (FTIR), 2D-IR, and vibrational circular dichroism spectra. As a result of their distinct β-sheet structure, 1–108-αS fibrils resist incorporation of WT-αS monomers.

2D IR spectroscopy of high-pressure phases of ice.

We present experimental and simulated 2D IR spectra of some high-pressure forms of isotope-pure D2O ice and compare the results to those of ice Ih published previously [F. Perakis and P. Hamm, Phys. Chem. Chem. Phys. 14, 6250 (2012); L. Shi et al., ibid. 18, 3772 (2016)]. Ice II, ice V, and ice XIII have been chosen for this study, since this selection covers many aspects of the polymorphism of ice. That is, ice II is a hydrogen-ordered phase of ice, in contrast to ice Ih, while ice V and ice XIII are a hydrogen-disordered/ordered couple that shares essentially the same oxygen structure and hydrogen-bonded network. For the transmission 2D IR spectroscopy, a novel method had to be developed for the preparation of ultrathin films (1-2 μm) of high-pressure ices with good optical quality. We also simulated 2D IR spectra based on molecular dynamics simulations connected to a vibrational exciton picture. These simulations agree with the experimental results in a semi-quantitative manner for ice II, while the same approach failed for ice V and ice XIII. From the perspective of 2D IR spectroscopy, ice II appears to be more inhomogeneously broadened than ice Ih, despite its hydrogen-order, which we attribute to the fact that ice II is structurally more complex with four distinguishable hydrogen bonds that mix due to exciton coupling. Ice V and ice XIII, on the other hand, behave as expected with the hydrogen-disordered case (ice V) being more inhomogenously broadened. Furthermore, in all hydrogen-ordered forms (ice II and ice XIII), cross peaks could be identified in the anisotropic 2D IR spectrum, whose signs reveal the relative direction of the corresponding excitonic states.

Efficient Vibrational Energy Transfer through Covalent Bond in Indigo Carmine Revealed by Nonlinear IR Spectroscopy.

Ultrafast vibrational relaxation and structural dynamics of indigo carmine in dimethyl sulfoxide were examined using femtosecond pump-probe infrared and two-dimensional infrared (2D IR) spectroscopies. Using the intramolecularly hydrogen-bonded C═O and delocalized C═C stretching modes as infrared probes, local structural and dynamical variations of this blue dye molecule were observed. Energy relaxation of the vibrationally excited C═O stretching mode was found to occur through covalent bond to the delocalized aromatic vibrational modes on the time scale of a few picoseconds or less. Vibrational quantum beating was observed in magic-angle pump-probe, anisotropy, and 2D IR cross-peak dynamics, showing an oscillation period of ca. 1010 fs, which corresponds to the energy difference between the C═O and C═C transition frequency (33 cm-1). This confirms a resonant vibrational energy transfer happened between the two vibrators. However, a more efficient energy-accepting mode of the excited C═O stretching was believed to be a nearby combination and/or overtone mode that is more tightly connected to the C═O species. On the structural aspect, dynamical-time-dependent 2D IR spectra reveal an insignificant inhomogeneous contribution to time-correlation relaxation for both the C═O and C═C stretching modes, which is in agreement with the generally believed structural rigidity of such conjugated molecules.