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The experimentally derived coupling model is used to simulate and interpret the absorption and vibrational circular dichroism (VCD) spectra of several model double-stranded nucleic acids in the region of the nucleobase carbonyl stretches. It was found that VCD and absorption spectra for all the oligomers, poly(dG)-poly(dC), poly(dG-dC)-and dGGCC can be simulated within the uncertainties of the coupling model.

2D IR spectroscopy and 1-13C=18O isotope labeling was used to measure the vibrational dynamics of 11 amide I modes in the CD3[zeta] transmembrane domain. It was found that the homogeneous line widths ad population relaxation times were all nearly identical but that amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane.

Heterodyned 2D-IR, frequency resolved photon echo, and pump - probe spectroscopies were collected to study the couplings and anharmonicities of cytidine and guanosine bases in DNA. Cross-peaks between the G and C carbonyl bands that are interpreted as interstrand coupling across the two oligomers is observed, and strong intrastrand coupling between stacked G carbonyls is also presented.

The two-dimensional infrared (2D IR) methods are used to determine the features of the vibrational dynamics and structure of the dipeptide acetylproline-NH2 in D2O and CDCl3. The result obtained from the measurements shows diagonal peaks and cross-peaks that are determined by the structures and vibrational dynamics of the acetylproline-NH2 molecule.

The application of DNA oligomers with pairs of nitrile groups to study distances and angles in dual labeled molecules is demonstrated. The small sizes of nitrile groups makes the DNA model better probes of sterically confined regions like the inner cores of large biomolecules, compared to other spectroscopic probes.

Two-dimensional infrared (2D IR) spectroscopy is applied to study the carbonyl vibrational modes of guanine and cytosine bases in A- and B-form DNA. The results have shown that the coupling is very strong for both hydrogen-bonded and stacked base geometries, creating vibrational modes that extend both across the base pairs and along the lengths of the helices.

We used two-dimensional IR bioimaging to study the structural heterogeneity of formalin-fixed mouse pancreas. Images were generated from the hyperspectral data sets by plotting quantities associated with the amide I vibrational mode, which is created by the backbone carbonyl stretch. Images that measure the fundamental vibrational frequencies, cross peaks, and anharmonic shifts are presented. Histograms are generated for each quantity, providing averaged values and distributions around the mean that serve as metrics for protein structures. Images were generated from tissue that had been stored in a formalin fixation for 3, 8, and 48 weeks. Over this period, all three metrics show that that the β-sheet content of the samples increased, consistent with protein aggregation. Our results indicate that formalin fixation does not entirely arrest the degradation of a protein structure in pancreas tissue.

Transition dipoles are an underutilized quantity for probing molecular structures. The transition dipole strengths in an extended system like a protein are modulated by the couplings and thus probe the structures. Here we measure the absolute transition dipole strengths of human and rat amylin in their solution, aggregated, membrane, and micelleular bound forms, using a combination of 1D and 2D infrared spectroscopy. We find that the vibrational modes of amyloid fibers made of human amylin can extend across as many as 12 amino acids, reflecting very ordered β-sheets in the most carefully prepared samples. Rat amylin has FTIR spectra that are nearly identical in solution, micelles, and membranes. We show that the transition dipoles of rat amylin are much larger when bound to micelles and membranes than when in solution, consistent with rat amylin adopting an α-helical structure. We interpret the transition dipole strengths as experimental measurements of the inverse participation ratio often calculated in theoretical studies. The structure of aggregating and membrane-bound proteins can be difficult to identify with existing techniques, especially during kinetics. These results demonstrate how absolute transition dipoles measured via our 1D/2D spectroscopy method can provide important structural information.

DNA-covered materials are important in technological applications such as biosensors and microarrays, but obtaining structural information on surface-bound biomolecules is experimentally challenging. In this paper, we structurally characterize single-stranded DNA monolayers of poly(thymine) from 10 to 25 bases in length with an emerging surface technique called two-dimensional sum frequency generation (2D SFG) spectroscopy. These experiments are carried out by adding a mid-IR pulse shaper to a femtosecond broad-band SFG spectrometer. Cross peaks and 2D line shapes in the 2D SFG spectra provide information about structure and dynamics. Because the 2D SFG spectra are heterodyne detected, the monolayer spectra can be directly compared to 2D infrared (2D IR) spectra of poly(thymine) in solution, which aids interpretation. We simulate the 2D SFG spectra using DFT calculations and an excitonic Hamiltonian that relates the molecular geometry to the vibrational coupling. Intrabase cross peaks help define the orientation of the bases and interbase cross peaks, created by coupling between bases, and resolves features not observed in 1D SFG spectra that constrain the relative geometries of stacked bases. We present a structure for the poly(T) oligomer that is consistent with the 2D SFG data. These experiments provide insight into the DNA monolayer structure and set precedent for studying complex biomolecules on surfaces with 2D SFG spectroscopy.

A high-sensitivity metal-carbonyl-based IR probe is described that can be incorporated into proteins or other biomolecules in very high yield via Click chemistry. A two-step strategy is demonstrated. First, a methionine auxotroph is used to incorporate the unnatural amino acid azidohomoalanine at high levels. Second, a tricarbonyl (η(5)-cyclopentadienyl) rhenium(I) probe modified with an alkynyl linkage is coupled via the Click reaction. We demonstrate these steps using the C-terminal domain of the ribosomal protein L9 as a model system. An overall incorporation level of 92% was obtained at residue 109, which is a surface-exposed residue. Incorporation of the probe into a surface site is shown not to perturb the stability or structure of the target protein. Metal carbonyls are known to be sensitive to solvation and protein electrostatics through vibrational lifetimes and frequency shifts. We report that the frequencies and lifetimes of this probe also depend on the isotopic composition of the solvent. Comparison of the lifetimes measured in H2O versus D2O provides a probe of solvent accessibility. The metal carbonyl probe reported here provides an easy and robust method to label very large proteins with an amino-acid-specific tag that is both environmentally sensitive and a very strong absorber.

A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

A series of non-natural infrared probes is reported that consist of a metal-tricarbonyl modified with a -(CH2)n- linker and cysteine-specific leaving group. They can be site-specifically attached to proteins using mutagenesis and similar protocols for EPR spin labels, which have the same leaving group. We characterize the label's frequencies and lifetimes using 2D IR spectroscopy in solvents of varying dielectric. The frequency range spans 10 cm(-1), and the variation in lifetimes ranges from 6 to 19 ps, indicating that these probes are very sensitive to their environments. Also, we attached probes with -(CH2)-, -(CH2)3-, and -(CH2)4- linkers to ubiquitin at positions 6 and 63 and collected spectra in aqueous buffer. The frequencies and lifetimes were correlated for 3C and 4C linkers, as they were in the solvents, but did not correlate for the 1C linker. We conclude that lifetime measures solvation, whereas frequency reflects the electrostatics of the environment, which in the case of the 1C linker is a measure of the protein electrostatic field. We also labeled V71C α-synuclein in buffer and membrane-bound. Unlike most other infrared labels, this label has extremely strong cross sections and thus can be measured with 2D IR spectroscopy at sub-millimolar concentrations. We expect that these labels will find use in studying the structure and dynamics of membrane-bound, aggregated, and kinetically evolving proteins for which high signal-to-noise at low protein concentrations is imperative.

Sum-frequency generation (SFG) spectroscopy is a ubiquitous tool in the surface sciences. It provides infrared transition frequencies and line shapes that probe the structure and environment of molecules at interfaces. In this article, we apply techniques learned from the multidimensional spectroscopy community to SFG spectroscopy. We implement balanced heterodyne detection to remove scatter and the local oscillator background. Heterodyning also separates the resonant and nonresonant signals by acquiring both the real and imaginary parts of the spectrum. We utilize mid-IR pulse shaping to control the phase and delay of the mid-IR pump pulse. Pulse shaping allows phase cycling for data collection in the rotating frame and additional background subtraction. We also demonstrate time-domain data collection, which is a Fourier transform technique, and has many advantages in signal throughput, frequency resolution, and line shape accuracy over existing frequency domain methods. To demonstrate time-domain SFG spectroscopy, we study an aryl isocyanide on gold, and find that the system has an inhomogeneous structural distribution, in agreement with computational results, but which was not resolved by previous frequency-domain SFG studies. The ability to rapidly and actively manipulate the mid-IR pulse in an SFG pules sequence makes possible new experiments and more accurate spectra., (© 2011 American Chemical Society)