Department of Chemistry, Tulane University, New Orleans, Louisiana 70118, USA.
Acc Chem Res. 2009 Sep 15;42(9):1385-94. doi: 10.1021/ar900008p.
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.
发展用于测量三维结构和结构变化动力学的新方法对于包括结构生物学在内的许多自然科学都很重要，这可以帮助我们理解分子识别和催化的物理基础。二维红外（2DIR）光谱方法可以测量分子中振动模式之间的成对相互作用，为提供结构约束提供分子尺度的标尺，例如振动模式之间的距离、它们的跃迁偶极子之间的角度以及它们之间的能量转移速率。虽然最近已经有很多系统使用 2DIR 进行了研究，但仍存在如何测量较大分子的结构特征的问题。与蛋白质等较大分子合作存在挑战，例如振动光谱非常拥挤、2DIR 方法可访问的距离范围很小以及灵敏度问题。本综述描述了我们实验室克服其中一些挑战的努力。首先，我们讨论了双频 2DIR 方法，该方法为特定的一对振动报告器提供最高的选择性和最高的灵敏度。其次，我们描述了开发新型 2DIR 振动标签的步骤，例如 C 相同 N 和 C-D 伸缩模式，其频率在水透明区域以及指纹区域内。讨论了适合标记氨基酸的方案。接下来，我们描述了我们实验室开发的新型弛豫辅助 2DIR（RA 2DIR）方法。该方法利用分子中的振动弛豫和振动能量输运以及分子尺度上的热化过程来产生更强的交叉峰。使用 RA 2DIR 方法观察到相隔约 11 A 的模式的 18 倍交叉峰放大，并且对于模式之间的较大距离预计会有更大的放大。RA 2DIR 方法提供的大放大增强了 2DIR 光谱的灵敏度，并允许进行更大范围的结构测量。除了产生更强的交叉峰外，还证明了能量输运时间与模式间距离之间的相关性。这种相关性允许在与总相关谱（TOCSY）和二维核磁共振（NMR）光谱的异核多键相关（HMBC）方法非常相似的分子中测量模式连接性模式。我们希望，通过适当的校准，RA 2DIR 方法将能够快速评估分子中的距离和键连接性模式，并达到分析方法的水平。
