Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA; Natural Science Division, Campbellsville University, Campbellsville, KY 42718, USA.
Biochim Biophys Acta Biomembr. 2022 Mar 1;1864(3):183836. doi: 10.1016/j.bbamem.2021.183836. Epub 2021 Dec 11.
There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S pinholin. This ESEEM approach uses a combination of site-directed spin labeling and H-labeled side chains. Pinholin S is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 3-helix which have been observed in other channel forming proteins.
最近，在使用电子顺磁共振（EPR）检测膜蛋白局部二级结构的方法上有了一些进展。本文采用三脉冲电子自旋回波包络调制（ESEEM）方法来确定小孔形成膜蛋白Spinholin 的局部螺旋二级结构。这种 ESEEM 方法结合了定点自旋标记和 H 标记侧链。Spinholin S 负责双链 DNA 噬菌体宿主细胞的内胞质膜的通透性。在这项研究中，我们报告了活性形式和阴性优势失活突变体形式的 Spinholin 的圆二色性（CD）光谱的整体全局螺旋结构。使用 ESEEM 光谱技术证实了活性和失活的 Spinholin 的两个跨膜结构域（TMD）的局部螺旋二级结构。每个跨膜结构域的 ESEEM 归一化频域强度的比较深入了解了这些结构域的α螺旋折叠性质，而不是在其他通道形成蛋白中观察到的π或 3 螺旋。
