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。由于其灵敏度和时间分辨实验的能力，从飞秒到小时的时间尺度振动光谱是一个理想的工具，研究这些蛋白质/蛋白质和蛋白质/辅因子的相互作用。然而，这种高度复杂的生物分子的振动光谱的解释，通常是复杂的强烈重叠的信号，导致一个模糊的分子分配。我们建议通过结合生物化学和光谱方法来解决这个瓶颈。在生物化学方面，我们将建立有效的位点特异性同位素标记协议的辅因子和蛋白质侧链。蛋白质样品将在工程化的E.缺乏选定的氨基酸或辅因子生物合成基因的大肠杆菌菌株，从而能够选择性地进料同位素标记的底物。然后通过振动（红外和拉曼散射）光谱法，使用具有ps（泵浦-探测）至min（步进-扫描）时间分辨率的最新方法，研究标记的蛋白质样品。作为一个模型系统，我们将研究BLUF（蓝光传感器使用FAD）感光域。它们包含一个迄今为止知之甚少的黄素辅因子，它被一个氢键网络包围，在闪光后的皮秒内可逆地转换。利用该模型系统，我们希望建立一个高效的，通用的方法来研究复杂的生物分子。为此，我们将联合收割机结合两个专业研究小组的专业知识，这两个研究小组在光感受器的生化和光谱研究方面具有丰富的经验。两个实验室之间的生物化学家和光谱学家的频繁交流加强了这种合作。

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.