Stucki-Buchli Brigitte, Johnson Philip J M, Bozovic Olga, Zanobini Claudio, Koziol Klemens L, Hamm Peter, Gulzar Adnan, Wolf Steffen, Buchenberg Sebastian, Stock Gerhard
Department of Chemistry, University of Zurich , 8075 Zurich, Switzerland.
Biomolecular Dynamics, Institute of Physics, Albert Ludwigs University , 79104 Freiburg, Germany.
J Phys Chem A. 2017 Dec 14;121(49):9435-9445. doi: 10.1021/acs.jpca.7b09675. Epub 2017 Dec 4.
We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensity change. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans-cis photoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before.
我们借助二维红外差分光谱，探究非天然氨基酸叠氮高丙氨酸（AHA）作为红外标签感知蛋白质中相对较小结构变化的能力。为此，我们用AHA标记了一种变构蛋白（人酪氨酸磷酸酶1E的PDZ2结构域），并进一步将其与一种偶氮苯衍生的光开关共价连接，以模拟其在配体结合时的构象转变。为了确定AHA标签的优势和局限性，我们总共研究了六个突变体，标签位于具有不同性质的位点。只有一个突变体显示出可测量的二维红外差分信号。与通常观察到的反映溶剂化程度的频率变化不同，在这种情况下我们观察到的是强度变化。为了理解这种光谱响应，我们进行了经典分子动力学模拟，评估AHA标签与水分子和蛋白质侧链的局部接触，并基于静电模型计算振动频率。尽管这些模拟部分揭示了反式-顺式光异构化过程中蛋白质内部和与水接触数量的显著而复杂的变化，但它们无法清楚解释为什么这个标签会突出。随后的量子化学计算表明，这种响应是一种电子相互作用的结果，涉及叠氮基团与光开关中磺酸基团的电荷转移。据我们所知，这种效应以前尚未被描述过。
