2D infrared (IR) spectroscopy is a cutting-edge technique, with applications in subjects as diverse as the energy sciences, biophysics and physical chemistry. This book introduces the essential concepts of 2D IR spectroscopy step-by-step to build an intuitive and in-depth understanding of the method. This unique book introduces the mathematical formalism in a simple manner, examines the design considerations for implementing the methods in the laboratory, and contains working computer code to simulate 2D IR spectra and exercises to illustrate involved concepts. Readers will learn how to accurately interpret 2D IR spectra, design their own spectrometer and invent their own pulse sequences. It is an excellent starting point for graduate students and researchers new to this exciting field. Computer codes and answers to the exercises can be downloaded from the authors' website, available at www.cambridge.org/9781107000056.

Femtosecond infrared (IR) pump probe and dynamic hole burning experiments were used to examine the ultrafast response of the modes in the 1600-1700 cm -1 region (the so-called amide I modes) of N-methylacetamide (NMA) and three small globular peptides, apamin, scyllatoxin, and bovine pancreatic trypsin inhibitor (BPTI). A value of 16 cm -1 was found for the anharmonicity of the amide I vibration. Vibrational relaxation of the amide I modes of all investigated peptides occurs in ca. 1.2 ps. An even faster value of 450 fs is obtained for NMA, a model for the peptide unit. The vibrational relaxation is dominated by intramolecular energy redistribution (IVR) and reflects an intrinsic property of the peptide group in any environment. Dynamic hole burning experiments with a narrow band pump pulse which selectively excites only a subset of the amide I eigenstates reveal that energy migration between different amide I states is slow compared with vibrational relaxation. Two-dimensional pump-probe (2D-IR) spectra that display the spectral response of the amide I band as a function of the frequency of the narrow band pump pulse show that the amide I states are nevertheless delocalized along the peptide backbone. A simple excitonic coupling model describes the nonlinear pump-probe spectrum, and it reproduces the experimental 2D-IR spectra. It is estimated that the accessible peptide excitons are delocalized over a length of ca. 8 Å.

Using polarization sensitive two-dimensional (2D) vibrational spectroscopy on the amide I mode, the central backbone structure of trialanine in aqueous solution is investigated. We exploit the polarization sensitivity of the 2D pump−probe signal to reveal the cross-peak structure hidden under the strong diagonal peaks. The dihedral angles φ and ψ characterizing the peptide backbone structure are derived directly from the cross-peak intensity and anisotropy, demonstrating the potential of 2D spectroscopy as a tool for peptide structure elucidation.

The vibrational cooling of azobenzene after photoisomerization is investigated by time resolved IR spectroscopy with femtosecond time resolution. Transient difference spectra were obtained in a frequency range where phenyl ring modes and the central N=N-stretching mode absorbs. The experimental data are discussed in terms of a simple theoretical model which was derived in order to account for the off-diagonal anharmonicity between the investigated high-frequency modes and the bath of the remaining low-frequency modes in a polyatomic molecule. It is shown that these off-diagonal anharmonic constants dominate the observed transient absorbance changes while the anharmonicity of the high-frequency modes themselves (diagonal anharmonicity) causes only minor effects. Based on the transient IR spectra, the energy flow in the azobenzene molecule can be described as follows: After an initial ultrafast intramolecular energy redistribution process, the decay of the related intramolecular temperature occurs via intermolecular energy transfer to the solvent on a time scale of ca. 20 ps.

A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology.The three-dimensional (3D) structure of peptides and proteins and their fluctuations are essential properties responsible for the extremely high specificity of biological reactions. Progress in understanding biological processes such as enzyme reactions originates from the detailed knowledge of the secondary, tertiary, and quarternary structures of the participating biomolecules. Two major spectroscopic techniques are responsible for this development: x-ray scattering, which maps out the electron density of the molecule, and two-dimensional (2D)-NMR spectroscopy (1-3), which can measure the distances between pairs of protons. The next step must be the determination of structures in motion over a wide range of time scales. We believe that multidimensional IR spectroscopy can address this new challenge.The IR spectra of the amide transitions provide information about secondary structural motifs of proteins and peptides. The so-called amide I band, which consists of mostly the stretching motion of the peptide backbone CϭO groups, is a strong IR absorber, which is spectrally isolated from other vibrational modes such as those from amino acid side groups. The amide I states can be viewed as vibrational excitons (4, 5) with the individual peptide groups considered as separated but coupled units. The coupling could be either through-space, such as multipole or even dipole-dipole interaction as proposed by Krimm and Bandekar (4), or through-bond, involving charge shifts and kinematic coupling via the C ␣ atoms of the backbone. The states, which have one excitation present in the whole assembly, can be interrogated by conventional (linear) IR absorption spectroscopy, but the information obtained is insufficient to determine the coupling Hamiltonian, from which a structure might be deduced. More information is available from nonlinear third-order spectroscopic techniques (6) such as the 2D experiment presented here. In a separated system picture both the one-exciton and two-exciton states contribute to the nonlinear IR signal in such a way that all couplings between the separate amide units are available, and in principle a 3D structure of the peptide can be deduced.The success of NMR sp...

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Nonlinear time-resolved vibrational spectroscopy is used to compare spectral broadening of the amide I band of the small peptide trialanine with that of N-methylacetamide, a commonly used model system for the peptide bond. In contrast to N-methylacetamide, the amide I band of trialanine is significantly inhomogeneously broadened. Employing classical molecular-dynamics simulations combined with density-functional-theory calculations, the origin of the spectral inhomogeneity is investigated. While both systems exhibit similar hydrogen-bonding dynamics, it is found that the conformational dynamics of trialanine causes a significant additional spectral broadening. In particular, transitions between the poly͑Gly͒II and the ␣ R conformations are identified as the main source of the additional spectral inhomogeneity of trialanine. The experimental and computational results suggest that trialanine adopts essentially two conformations: poly͑Gly͒II ͑80%͒ and ␣ R ͑20%͒. The potential of the joint experimental and computational approach to explore conformational dynamics of peptides is discussed.

X-ray crystallography and nuclear magnetic resonance measurements provide us with atomically resolved structures of an ever-growing number of biomolecules. These static structural snapshots are important to our understanding of biomolecular function, but real biomolecules are dynamic entities that often exploit conformational changes and transient molecular interactions to perform their tasks. Nuclear magnetic resonance methods can follow such structural changes, but only on millisecond timescales under non-equilibrium conditions. Time-resolved X-ray crystallography has recently been used to monitor the photodissociation of CO from myoglobin on a subnanosecond timescale, yet remains challenging to apply more widely. In contrast, two-dimensional infrared spectroscopy, which maps vibrational coupling between molecular groups and hence their relative positions and orientations, is now routinely used to study equilibrium processes on picosecond timescales. Here we show that the extension of this method into the non-equilibrium regime allows us to observe in real time in a short peptide the weakening of an intramolecular hydrogen bond and concomitant opening of a beta-turn. We find that the rate of this process is two orders of magnitude faster than the 'folding speed limit' established for contact formation between protein side chains.