An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron–electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.

中文翻译：

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电子顺磁共振波谱研究的天然和熔融小球状态麦芽糖结合蛋白的打开和关闭形式

深入研究的蛋白质折叠的中间状态是熔融小球（MG）状态，该状态包含二级但几乎没有任何三级结构。在先前的工作中，我们使用连续波和双电子-电子共振（DEER）电子顺磁共振（EPR）光谱法确定了处于天然状态的麦芽糖结合蛋白（MBP）中相互作用的自旋之间的距离。已经使用七个双突变体来研究MBP的两个结构域内的结构。DEER数据很好地证实了先前可用的X射线数据。即使处于MG状态，MBP仍会结合其配体麦芽糖。因此，我们假设在不存在三级结构的情况下，MBP的结合口袋周围必须已经有一个确定的结构。在这里，我们研究了一组新的MBP突变体在开放和封闭状态下天然状态与MG状态之间的功能和结构差异。在这些标记中，自旋标记位置位于活性位点附近。其配体的结合导致从开放状态到封闭状态的构象变化，其中两个结构域更紧密地结合在一起。使用双量子相干EPR在pH 3.2（MG）和pH 7.4（天然状态）下分析了完整的MBP突变体。将该值与通过分子建模从开式和闭式MBP的晶体结构通过分子建模构建的双基标记之间的距离的理论预测值进行比较，发现它们之间具有极好的一致性。测量结果显示，MG中MBP的结合口袋周围有确定的结构，这解释了麦芽糖的结合。