Relaxation-assisted two-dimensional infrared (RA 2DIR) method: accessing distances over 10 A and measuring bond connectivity patterns.

Development of new approaches for measuring three-dimensional structures and dynamics of structural changes is important for a number of natural sciences, including structural biology, where it can lead to understanding the physical bases of molecular recognition and catalysis. A two-dimensional infrared (2DIR) spectroscopy method permits measuring pairwise interactions among vibrational modes in molecules providing a molecular scale ruler for delivering structural constraints, such as the distances between the vibrational modes, angles between their transition dipoles, and the energy-transfer rates between them. While there is a large variety of systems that have recently been interrogated using 2DIR, questions remain of how to measure structural features of larger molecules. The challenges of working with larger molecules, such as proteins, include very congested vibrational spectra, a small range of distances accessible by the 2DIR method, and sensitivity issues. This Account describes the efforts of our laboratory to overcome some of these challenges. First, we discuss the dual-frequency 2DIR approach, which provides the highest selectivity to a particular pair of vibrational reporters and highest sensitivity. Second, we describe our steps in developing vibrational labels, novel for 2DIR, such as C identical withN and C-D stretching modes that have frequencies in the water transparency region, as well as the modes in the fingerprint region. The schemes suitable for labeling amino acids are discussed. Next, we describe the novel relaxation-assisted 2DIR (RA 2DIR) method, developed in our laboratory. The method uses vibrational relaxation and vibrational energy transport in molecules and the thermalization process on a molecular scale, to generate stronger cross-peaks. An 18-fold cross-peak amplification was observed for the modes separated by about 11 A using the RA 2DIR method, and larger amplifications are expected for larger distances between the modes. Large amplification provided by the RA 2DIR method enhances the sensitivity of 2DIR spectroscopy and permits longer range structural measurements. In addition to generating stronger cross-peaks, a correlation of the energy transport time with the intermode distance is demonstrated. This correlation permits measurements of mode-connectivity patterns in molecules much similar to those available in total correlation spectroscopy (TOCSY) and heteronuclear multiple-bond correlation (HMBC) methods of 2D nuclear magnetic resonance (NMR) spectroscopy. It is our hope that, with a proper calibration, the RA 2DIR method will permit speedy assessments of distances and the bond connectivity patterns in molecules and reach the level of an analytical method.