Two-dimensional IR spectroscopy can be designed to eliminate the diagonal peaks and expose only the crosspeaks needed for structure determination

Abstract

The power of two-dimensional (2D) IR spectroscopy as a structural method with unprecedented time resolution is greatly improved by the introduction of IR polarization conditions that completely eliminate diagonal peaks from the spectra and leave only the crosspeaks needed for structure determination. This approach represents a key step forward in the applications of 2D IR to proteins, peptides, and other complex molecules where crosspeaks are often obscured by diagonal peaks. The technique is verified on the model compound 1,3-cyclohexanedione and subsequently used to clarify the distribution of structures that the acetylproline-NH 2 dipeptide adopts in chloroform. In both cases, crosspeaks are revealed that were not observed before, which, in the case of the dipeptide, has led to additional information about the structure of the amino group end of the peptide.H eterodyned 2D IR experiments have recently been reported that are the IR analogues of pulsed correlated spectroscopy and nuclear Overhauser effect spectroscopy in 2D NMR (1-5). Like 2D NMR spectra that exhibit diagonal peaks at frequencies determined by one-dimensional spectra and crosspeaks between coupled nuclear spins, 2D IR spectra contain diagonal peaks and crosspeaks that represent coupled vibrational transitions. The 2D IR crosspeak intensities and splittings depend on the angles and distances between the vibrational modes and can be used to characterize the structures of peptides (3, 6, 7). One advantage (8) of 2D IR over other structure-determining methods is the time scale; 2D IR can monitor structures on a picosecond timescale, which allows transient protein dynamics to be followed. However, structural information is lost when the crosspeaks overlap with the more intense diagonal peaks. Methods have been developed in 2D NMR to minimize the overlap of diagonal and crosspeaks (9, 10). Here we report a technique based on quite different principles that completely eliminates the diagonal peaks from 2D IR spectra and allows the crosspeaks and hence structures to be more easily and better characterized.A useful characteristic of IR transitions is that the directions of their transition dipoles in the molecular frame are often predictable. For example, modes that are mainly localized on two atoms such as NOH, COH, or CAO stretches usually have transition dipole vectors near the bond axis, and the amide I modes of peptide units have transition dipoles whose directions are given by the positions of the amide atoms (11). The four polarized IR pulses in the 2D IR experiments successively interact with four transition dipoles of each molecule, and thus polarized 2D IR measurements can yield information on the geometric arrangements of the dipoles. The diagonal peaks are generated when the IR pulses all interact with the same mode and therefore interrogate only a single location in the structure. The crosspeaks are generated when the IR pulses interact with modes in different locations. The results are couplings and relative orientations that can be used t...

Spectroscopic elucidation of uncoupled transition energies in the major photosynthetic light-harvesting complex, LHCII

“…1 displays absolute value, nonrephasing spectra of the Q y region of the chlorophyll in LHCII under two different polarization sequences of the incident laser fields, with all-parallel (0, 0, 0, 0) on the left and "cross-peak-specific" (π/3, −π/3, 0, 0) on the right (10,24). Under the cross-peak-specific polarization, the signal along the diagonal corresponding to peaks in the linear absorption spectrum is removed and the relative amplitudes of off-diagonal features change (10,18,24). On the 2D spectra, tick marks divide three regions: (i) Chla (14,700 cm −1 -15,000 cm −1 ), with a low-and midenergy band; (ii) intermediate (15,000 cm −1 -15,200 cm −1 ), with high-energy Chla and low-energy Chlb; and (iii) Chlb (15,200 cm −1 -15,500 cm −1 ).…”

Cross-peak-specific two-dimensional electronic spectroscopy

“…This figure includes information for excited-state absorption and relaxation pathways not shown by Dreyer et al (7), and we have corrected two errors in their table. These factors provide a key for inferring structural information from 2D spectra in the form of projection angles between transition dipole moments, as demonstrated by several groups in the IR (16)(17)(18). Additionally, the signs corresponding to the orientational factors for a given pathway allow us to assign spectral features, because in general pathways carry a certain sign regardless of the angle between coupled transitions (although R* 1 (ij) and coherence transfer pathways, which are not considered here, can be either positive or negative).…”

2D IR Spectroscopy with Phase-Locked Pulse Pairs from a Birefringent Delay Line

“…It is therefore particularly well suited for anisotropy measurements and signal amplification using polarizers. This can also be exploited to determine angles between transition dipole moments with improved accuracy [13] and to eliminate diagonal peaks by subtracting signals recorded with polarization orders Y XY X and XYY X [18]. The exceptional accuracy in setting the t 1 delay could also be exploited for accurate and fast undersampling of the t1-axis and improve speed of acquisition in the case of step scan acquisition.…”

“…They can be projected to the same polarization with an additional polarizer for conventional 2D spectroscopy [17]. However, it is also possible to use the two perpendicularly polarized pulses to remove scattering and enhance signal with the use of polarizers in the probe beam [13,14,18,19].…”