International Tomography Center SB RAS, 630090 Novosibirsk, Russia.
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia; Novosibirsk State University, Pirogova Str. 2, Novosibirsk 630090, Russia.
J Photochem Photobiol B. 2020 Oct;211:112008. doi: 10.1016/j.jphotobiol.2020.112008. Epub 2020 Aug 27.
Recently, a new type of spin labels based on photoexcited triplet molecules was proposed for nanometer scale distance measurements by pulsed dipolar electron paramagnetic resonance (PD EPR). However, such molecules are also actively used within biological complexes as photosensitizers for photodynamic therapy (PDT) of cancer. Up to date, the idea of using the photoexcited triplets simultaneously as PDT agents and as spin labels for PD EPR has never been employed. In this work, we demonstrate that PD EPR in conjunction with other methods provides valuable information on the structure and function of PDT candidate complexes, exemplified here with porphyrins bound to human serum albumin (HSA). Two distinct porphyrins with different properties were used: amphiphilic meso-tetrakis(4-hydroxyphenyl)porphyrin (mTHPP) and water soluble cationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4); HSA was singly nitroxide-labeled to provide a second tag for PD EPR measurements. We found that TMPyP4 locates in a cavity at the center of the four-helix bundle of HSA subdomain IB, close to the interface with solvent, thus being readily accessible to oxygen. As a result, the photolysis of the complex leads to photooxidation of HSA by generated singlet oxygen and causes structural perturbation of the protein. Contrary, in case of mTHPP porphyrin, the binding occurs at the proton-rich pocket of HSA subdomain IIIA, where the access of oxygen to a photosensitizer is hindered. Structural data of PD EPR were supported by other EPR techniques, laser flash photolysis and protein photocleavage studies. Therefore, pulsed EPR on complexes of proteins with photoexcited triplets is a promising approach for gaining structural and functional insights into such PDT agents.
最近，一种基于光激发三重态分子的新型自旋标记物被提出，可用于通过脉冲偶极电子顺磁共振（PD EPR）进行纳米级距离测量。然而，此类分子也在生物复合物中被积极用作光动力疗法（PDT）的光敏剂来治疗癌症。迄今为止，从未有人将光激发三重态同时用作 PDT 试剂和 PD EPR 的自旋标记物的想法。在这项工作中，我们证明了 PD EPR 与其他方法结合使用可为 PDT 候选复合物的结构和功能提供有价值的信息，这里以与人血清白蛋白（HSA）结合的卟啉为例。使用了两种具有不同性质的不同卟啉：两亲性的meso-四（4-羟苯基）卟啉（mTHPP）和水溶性的阳离子meso-四（N-甲基-4-吡啶基）卟啉（TMPyP4）；HSA 被单一的氮氧自由基标记，为 PD EPR 测量提供了第二个标记。我们发现 TMPyP4 位于 HSA 亚结构域 IB 的四螺旋束中心的腔体内，靠近与溶剂的界面，因此很容易与氧气接触。结果，该复合物的光解导致由生成的单线态氧对 HSA 的光氧化，并引起蛋白质的结构扰动。相反，对于 mTHPP 卟啉，结合发生在 HSA 亚结构域 IIIA 的质子丰富口袋中，其中氧气进入光敏剂受到阻碍。PD EPR 的结构数据得到了其他 EPR 技术、激光闪光光解和蛋白质光裂解研究的支持。因此，用光激发三重态的蛋白质复合物进行脉冲 EPR 是一种很有前途的方法，可用于深入了解此类 PDT 试剂的结构和功能。
