Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Acc Chem Res. 2012 Nov 20;45(11):1866-74. doi: 10.1021/ar200275k. Epub 2012 Mar 20.
Proteins, enzymes, and other biological molecules undergo structural dynamics as an intrinsic part of their biological functions. While many biological processes occur on the millisecond, second, and even longer time scales, the fundamental structural dynamics that eventually give rise to such processes occur on much faster time scales. Many decades ago, chemical kineticists focused on the inverse of the reaction rate constant as the important time scale for a chemical reaction. However, through transition state theory and a vast amount of experimental evidence, we now know that the key events in a chemical reaction can involve structural fluctuations that take a system of reactants to its transition state, the crossing of a barrier, and the eventual relaxation to product states. Such dynamics occur on very fast time scales. Today researchers would like to investigate the fast structural fluctuations of biological molecules to gain an understanding of how biological processes proceed from simple structural changes in biomolecules to the final, complex biological function. The study of the fast structural dynamics of biological molecules requires experiments that operate on the appropriate time scales, and in this Account, we discuss the application of ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy to the study of protein dynamics. The 2D IR vibrational echo experiment is akin to 2D NMR, but it operates on time scales many orders of magnitude faster. In the experiments, a particular vibrational oscillator serves as a vibrational dynamics probe. As the structure of the protein evolves in time, the structural changes are manifested as time-dependent changes in the frequency of the vibrational dynamics probe. The 2D IR vibrational echo experiments can track the vibrational frequency evolution, which we then relate to the time evolution of the protein structure. In particular, we measured protein substate interconversion for mutants of myoglobin using 2D IR chemical exchange spectroscopy and observed well-defined substate interconversion on a sub-100 ps time scale. In another study, we investigated the influence of binding five different substrates to the enzyme cytochrome P450(cam). The various substrates affect the enzyme dynamics differently, and the observed dynamics are correlated with the enzyme's selectivity of hydroxylation of the substrates and with the substrate binding affinity.
蛋白质、酶和其他生物分子的结构动力学是其生物功能的固有组成部分。虽然许多生物过程发生在毫秒、秒甚至更长的时间尺度内，但最终导致这些过程的基本结构动力学发生在更快的时间尺度内。几十年前，化学动力学家关注的是反应速率常数的倒数作为化学反应的重要时间尺度。然而，通过过渡态理论和大量的实验证据，我们现在知道化学反应中的关键事件可以涉及结构波动，这些波动将反应物系统带到其过渡态，跨越势垒，最终弛豫到产物状态。这种动力学发生在非常快的时间尺度上。如今，研究人员希望研究生物分子的快速结构波动，以了解生物过程如何从生物分子的简单结构变化发展到最终的复杂生物功能。生物分子快速结构动力学的研究需要在适当的时间尺度上进行实验，在本报告中，我们讨论了超快二维红外（2D IR）振动回声光谱在蛋白质动力学研究中的应用。2D IR 振动回声实验类似于 2D NMR，但它在许多数量级更快的时间尺度上运行。在实验中，特定的振动振荡器用作振动动力学探针。随着蛋白质结构随时间的演变，结构变化表现为振动动力学探针频率随时间的变化。2D IR 振动回声实验可以跟踪振动频率的演变，我们将其与蛋白质结构的时间演变联系起来。特别是，我们使用 2D IR 化学交换光谱法测量肌红蛋白突变体的蛋白质亚基转换，在亚 100 ps 的时间尺度上观察到了明确的亚基转换。在另一项研究中，我们研究了将五种不同的底物结合到酶细胞色素 P450（cam）上对酶动力学的影响。不同的底物对酶动力学的影响不同，观察到的动力学与酶对底物羟化的选择性以及底物结合亲和力相关。
