Ultrafast dynamics of biomolecules studied by vibrational spectroscopy on selectively isotope labeled proteins

非共价相互作用，如氢键和偶极相互作用决定了所有的生物过程。由于其灵敏度和在飞秒到小时的时间尺度上进行时间分辨实验的能力，振动光谱学是研究这些蛋白质/蛋白质和蛋白质/辅因子相互作用的理想工具。然而，对这种高度复杂的生物分子的振动谱的解释通常由于强烈的重叠信号导致分子分配不明确而变得复杂。我们建议通过结合生化和光谱的方法来解决这一瓶颈。在生物化学方面，我们将为辅助因子和蛋白质侧链建立有效的位点特异性同位素标记方案。蛋白质样品将在缺乏选定氨基酸或辅因子生物合成基因的工程大肠杆菌菌株中生产，从而能够选择性地喂养同位素标记的底物。标记的蛋白质样品将通过振动（红外和拉曼散射）光谱研究，使用最先进的方法，ps（泵浦探针）到min（步进扫描）时间分辨率。作为一个模型系统，我们将研究BLUF（使用FAD的蓝光传感器）光感受器域。它们含有一种迄今为止尚不为人所知的黄素辅因子，它被一个氢键网络所包围，在闪光后的皮秒内可逆地切换。利用该模型系统，我们希望建立一种高效、通用的方法来研究复杂的生物分子。为此，我们将结合两个专业研究小组的专业知识，这两个小组在光感受器的生化和光谱研究方面具有丰富的经验。这一合作由于两个实验室之间的生化学家和光谱学家的频繁交流而得到加强。

Non-covalent interactions like hydrogen bonding and diploar interactions determine all biological processes. Because of its sensitivity and the ability to perform time resolved experiments on timescales from femtoseconds to hours vibrational spectroscopy is an ideal tool to study these protein/protein and protein/cofactor interactions. Interpretation of vibrational spectra of such highly complex biomolecules, however, is usually complicated by strongly overlapping signals leading to an ambigous molecular assignment. We propose to address this bottleneck by a combined biochemical and spectroscopic approach. On the biochemistry side we will establish efficient site specific isotope labeling protocols for both cofactor and protein side chains. The protein samples will be produced in engineered E. coli strains that lack selected amino acid or cofactor biosynthesis genes thus enabling to selectively feed isotope labeled substrates. The labeled protein samples will then be studied by vibrational (infrared and Raman scattering) spectroscopy using state-of-the-art methods with ps (pump-probe) to min (step-scan) time resolution. As a model system we will investigate BLUF (Blue light sensors using FAD) photoreceptor domains. They contain a so far poorly understood flavin cofactor, which is surrounded by a hydrogen bonded network that is reversibly switched within picoseconds after a light flash. Using this model system we want to establish a highly efficient, general method for studying complex biomolecules. To do so we will combine the expertise of two specialized research groups, which have profound experience on the biochemical and spectroscopic study of photoreceptors. This collaboration is intensified by frequent exchange of the biochemists and spectroscopists between the two laboratories.