Amide I Two-Dimensional Infrared Spectroscopy of Proteins

Abstract

We review two-dimensional infrared (2D IR) spectroscopy of the amide I protein backbone vibration. Amide I modes are known for secondary structural sensitivity derived from their protein-wide delocalization. However, amide I FTIR spectra often display little variation for different proteins due to the broad and featureless line shape that arises from different structural motifs. 2D IR offers increased structural resolution by spreading the spectra over a second frequency dimension to reveal two-dimensional line shapes and cross-peaks. In addition, it carries picosecond time resolution, making it an excellent choice for understanding protein dynamics. In 2D IR spectra, cross peaks arise from anharmonic coupling between vibrations. For example, the spectra of ordered antiparallel beta sheets shows a cross peak between the strong nu perpendicular mode at approximately 1620 cm(-1) and the weaker nu parallel mode at approximately 1680 cm(-1). In proteins with beta-sheet content, disorder spreads the cross peaks into ridges, which gives rise to a "Z"-shaped contour profile. 2D IR spectra of alpha helices show a flattened "figure-8" line shape, and random coils give rise to unstructured, diagonally elongated bands. A distinguishing quality of 2D IR is the availability of accurate structure-based models to calculate spectra from atomistic structures and MD simulations. The amide I region is relatively isolated from other protein vibrations, which allows the spectra to be described by coupled anharmonic local amide I vibrations at each peptide unit. One of the most exciting applications of 2D IR is to study protein unfolding dynamics. While 2D IR has been used to study equilibrium structural changes, it has the time resolution to probe all changes resulting from photoinitiated dynamics. Transient 2D IR has been used to probe downhill protein unfolding and hydrogen bond dynamics in peptides. Because 2D IR spectra can be calculated from folding MD simulations, opportunities arise for making rigorous connections. By introduction of isotope labels, amide I 2D IR spectra can probe site-specific structure with picosecond time resolution. This has been used to reveal local information about picosecond fluctuations and disorder in beta hairpins and peptides. Multimode 2D IR spectroscopy has been used to correlate the structure sensitivity of amide I with amide II to report on solvent accessibility and structural stability in proteins.

Molecular dynamics simulation exploration of unfolding and refolding of a ten-amino acid miniprotein

“…It enables the characterization of the mechanical response of protein at the nanometer scale (Sotomayor and Schulten 2007;Best et al 2003). Moreover, two-dimensional infrared (2D IR) spectroscopy, which maps vibrational coupling between molecular groups, can be used to probe protein folding and unfolding dynamics with picosecond time resolution (Golonzka et al 2001;Hunt 2009;Kolano et al 2006;Ganim et al 2008). Hydrogen-exchange mass-spectrometry can track the formation of secondary structures during folding by measuring how easily protons (H ?…”

Transient 2D-IR spectroscopy of inorganic excited states

“…Owing to the availability of a number of articles, reviews and, recently, a textbook that discuss the methodology in detail, the intention here is to focus on the spectroscopy and the information content of the data obtained. [11][12][13][14][15] A 2D-IR spectrum is obtained from a 3 rd order nonlinear optical process in which three resonant interactions occur between ultrashort infrared laser pulses and vibrational transitions of the sample. A diagram of the various pulse sequences discussed below is given in Figure 1 where the IR interactions are numbered 1-3 in temporal order.…”

“…These off-diagonal peaks arising from vibrational coupling can be applied to decipher spectral contributions in mixtures or to aid spectral assignment. [11][12][13][14][15] In addition, structural information on the studied molecule can be obtained from the 2D-IR spectrum. This is achieved through monitoring the relative amplitudes of diagonal and off-diagonal features with parallel and perpendicular polarization relationships between the pump (denoted 1&2 in Figs 1 and 2) and probe (3) pulses.…”

“…Finally, spectroscopies that provide the ability to correlate vibrational modes in ground and excited states will provide useful information on the molecular changes caused by excitation and the coupling of electronic and vibrational degrees of freedom. One approach to achieving these aims is to extend the multidimensional infrared spectroscopy methods that have found increasing application in the ground state, [11][12][13][14][15] to electronically-excited states. Two-dimensional infrared (2D-IR) spectroscopy, first implemented in 1998, 16 has its basis in the multidimensional NMR experiments that have made major contributions to structural biology research in recent years.…”

“…2D-IR is now employed extensively in the study of ground state systems. [11][12][13][14][15] Exemplars of this include the analysis of offdiagonal peak patterns to determine molecular geometries, 18,19 deconvolute complex spectra 20 or reveal vibrational 21,22 or chemical dynamics, 23,24 while 2D-IR lineshapes report on the fluctuations in a system introduced by interaction with its surroundings, typically nearby solvent molecules. [25][26][27] Crucially, much of this information is either impossible or technologically challenging to extract from 1D experiments.…”