In memory of the 85th birthday of Yakov S. Lebedev (Moscow), who died in 1996, we start this Review on soft-glass matrix effects in donor–acceptor complexes with an appreciation of his pioneering work on high-field EPR spectroscopy on tribochemically generated donor–acceptor complexes. The mechanochemical activation of polycrystalline mixtures of porphyrins (and other donors) and quinone acceptors was found to produce large concentrations of triplet donor molecules and donor–acceptor radical pairs with unusual stability. The Review is continued with reporting on W-band high-field EPR and fast-laser studies on disaccharide matrix effects on structure and dynamics of donor–acceptor protein complexes related to photosynthesis, including the non-oxygenic bacterial reaction center (RC) and the oxygenic RCs Photosystem I (PS I) and Photosystem II (PS II, preliminary results). Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, in particular trehalose and sucrose. Trehalose is most effective in protecting biostructures, both in vivo and in vitro. To clarify the molecular mechanisms of disaccharide bioprotection, structure and dynamics of sucrose and trehalose matrices at different controlled hydration levels were probed by perdeuterated nitroxide spin labels and native cofactor intermediates in their charge-separated states. Trehalose forms a homogeneous amorphous phase in which the hosted molecules are uniformly distributed. Notably, their rotational mobility at room temperature is dramatically impaired by the trehalose H-bonding network confinement to an extent that in normal protein–matrix systems is only observed at low temperatures around 150 K. From the experimental results, formation of an extended H-bonding network of trehalose with protein molecules is inferred, involving both bulk and local water molecules. The H-bond network extends homogeneously over the whole matrix integrating and immobilizing the hosted protein. Taken together, these observations suggest that in photosystems, such as bacterial RCs and PS I complexes, of different size and complexity regarding subunit composition and oligomeric organization, the molecular configuration of the cofactors involved in the primary processes of charge separation is not significantly distorted by incorporation into trehalose glass, even under extensive dehydration. By means of pulsed W-band high-field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, in conjunction with using isotope labeled water (D2O and H217O), the biologically important issue of sensing and quantification of local water in proteins is addressed. The bacterial RC embedded into the trehalose glass matrix is used as model system. The two native radical cofactor ions of the primary electron donor and acceptor as well as an artificial nitroxide spin label site-specifically attached to the protein surface are studied in the experiments. The three paramagnetic reporter groups probe distinctly different local environments. They sense water molecules via their magnetic hyperfine and quadrupole interactions with either deuterons or 17O nuclei. It is shown that by using oxygen-17 labeled water, quantitative conclusions can be drawn differentiating between local and bulk water. It is concluded that dry trehalose operates as anhydrobiotic protein stabilizer by means of selective changes in the first solvation shell of the protein upon trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding network. Such changes usually have an impact on the global function of a biological system. Finally, preliminary results of optical and W-band EPR experiments on the extremolytes ectoine and its derivative hydroxyectoine are reported; these compounds appear to share several stress-protecting properties with trehalose in terms of stabilizing protein matrices. For instance, they display remarkable stabilizing capabilities towards sensitive proteins and enzymes with respect to freeze-thawing, heat-treatment, and freeze-drying procedures. Moreover, hydroxyectoine is a good glass-forming compound and exhibits a remarkable bioprotective effect against desiccation and heat denaturation of functional protein complexes.

中文翻译：

---

脱水海藻糖基质对膜蛋白的软动态限制：高场 EPR 和快速激光研究

为纪念 1996 年去世的 Yakov S. Lebedev（莫斯科）85 岁生日，我们开始这篇关于供体 - 受体复合物中软玻璃基质效应的评论，以赞赏他在摩擦化学高场 EPR 光谱方面的开创性工作生成供体-受体复合物。发现卟啉（和其他供体）和醌受体的多晶混合物的机械化学活化产生高浓度的三线态供体分子和具有异常稳定性的供体 - 受体自由基对。该评论继续报告 W 波段高场 EPR 和快速激光研究双糖基质对与光合作用相关的供体 - 受体蛋白复合物的结构和动力学的影响，包括无氧细菌反应中心 (RC) 和含氧 RCs 光系统 I (PS I) 和光系统 II (PS II, 初步结果）。一些生物体通过采用无水生物状态，其中细胞内培养基含有大量二糖，特别是海藻糖和蔗糖，可以在完全脱水和高温下存活。海藻糖在体内和体外保护生物结构方面最为有效。为了阐明二糖生物保护的分子机制，在不同受控水合水平下蔗糖和海藻糖基质的结构和动力学通过过氘氮氧化物自旋标记和处于电荷分离状态的天然辅因子中间体进行了探测。海藻糖形成均匀的无定形相，其中宿主分子均匀分布。尤其，它们在室温下的旋转流动性受到海藻糖 H 键网络限制的显着影响，其程度在正常蛋白质-基质系统中仅在 150 K 左右的低温下观察到。 从实验结果来看，扩展的 H 键网络的形成推断海藻糖与蛋白质分子的结合，涉及大量和局部水分子。H 键网络在整个基质上均匀延伸，整合和固定宿主蛋白质。综上所述，这些观察结果表明，在光系统中，例如细菌 RC 和 PS I 复合物，在亚基组成和寡聚组织方面具有不同大小和复杂性，即使在严重脱水的情况下，参与电荷分离主要过程的辅因子的分子构型也不会因掺入海藻糖玻璃而显着扭曲。通过脉冲 W 波段高场多共振 EPR 光谱，例如 ELDOR 检测的 NMR 和 ENDOR，结合使用同位素标记的水（D2O 和 H217O），蛋白质中局部水的传感和量化的生物学重要问题是解决。嵌入海藻糖玻璃基质中的细菌 RC 用作模型系统。在实验中研究了主要电子供体和受体的两个天然自由基辅助因子离子以及人工氮氧化物自旋标记位点特异性地附着在蛋白质表面。三个顺磁报告基团探测明显不同的局部环境。它们通过与氘核或 17O 原子核的磁性超精细和四极相互作用来感知水分子。结果表明，通过使用氧 17 标记的水，可以得出区分局部水和散装水的定量结论。得出的结论是，干海藻糖通过海藻糖-基质脱水后蛋白质的第一个溶剂化壳的选择性变化以及随后氢键网络的变化而起到脱水生物蛋白质稳定剂的作用。这种变化通常会对生物系统的整体功能产生影响。最后，报告了极端分子四氢嘧啶及其衍生物羟基四氢嘧啶的光学和 W 波段 EPR 实验的初步结果；在稳定蛋白质基质方面，这些化合物似乎与海藻糖具有多种应激保护特性。例如，它们在冻融、热处理和冷冻干燥过程中对敏感蛋白质和酶表现出显着的稳定能力。此外，羟基四氢嘧啶是一种良好的玻璃形成化合物，对功能性蛋白质复合物的干燥和热变性具有显着的生物保护作用。