Cellular membranes exhibit a huge diversity of lipids and membrane proteins that differ in their properties and chemical structure. Cells organize these molecules into distinct membrane compartments characterized by specific lipid profiles and hydrophobic thicknesses of the respective domains. If a hydrophobic mismatch occurs between a membrane protein and the surrounding lipids, there can be functional consequences such as reduced protein activity. This phenomenon has been extensively studied for single-pass transmembrane proteins, rhodopsin, and small polypeptides such as gramicidin. Here, we investigate the E. coli rhomboid intramembrane protease GlpG as a model to systematically explore the impact of membrane thickness on GlpG activity. We used fully saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) model lipids and altered membrane thickness by varying the cholesterol content. Physical membrane parameters were determined by 2H and 31P NMR spectroscopy and correlated with GlpG activity measurements in the respective host membranes. Differences in bulk and annular lipids as well as alterations in protein structure in the respective host membranes were determined using molecular dynamics simulations. Our findings indicate that GlpG can influence the membrane thickness in DLPC/cholesterol membranes but not in DMPC/cholesterol membranes. Moreover, we observe that GlpG protease activity is reduced in DLPC membranes at low cholesterol content, which was not observed for DMPC. While a change in GlpG activity can already be due to smallest differences in the lipid environment, potentially enabling allosteric regulation of intramembrane proteolysis, there is no overall correlation to cholesterol-mediated lipid bilayer organization and phase behavior. Additional factors such as the influence of cholesterol on membrane bending rigidity and curvature energy need to be considered. In conclusion, the functionality of α-helical membrane proteins such as GlpG relies not only on hydrophobic matching but also on other membrane properties, specific lipid interaction, and the composition of the annular layer.

中文翻译：

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评估膜厚度对膜内蛋白酶 GlpG 功能的影响


细胞膜表现出种类繁多的脂质和膜蛋白，它们的特性和化学结构各不相同。细胞将这些分子组织成不同的膜区室，其特征是各个结构域的特定脂质谱和疏水厚度。如果膜蛋白与周围脂质之间发生疏水错配，则可能会产生功能后果，例如蛋白质活性降低。这种现象已针对单通道跨膜蛋白、视紫红质和小多肽（如短杆菌素）进行了广泛研究。在这里，我们研究了 大肠杆菌菱形膜内蛋白酶 GlpG 作为模型，以系统地探索膜厚度对 GlpG 活性的影响。我们使用全饱和的 1,2-二月桂酰-sn-甘油-3-磷酸胆碱 （DLPC） 和 1,2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱 （DMPC） 模型脂质，并通过改变胆固醇含量来改变膜厚度。物理膜参数通过 2H 和 31P NMR 波谱确定，并与相应宿主膜中的 GlpG 活性测量值相关。使用分子动力学模拟确定了体脂和环状脂质的差异以及各自宿主膜中蛋白质结构的改变。我们的研究结果表明，GlpG 可以影响 DLPC/胆固醇膜的膜厚度，但不会影响 DMPC/胆固醇膜的膜厚度。此外，我们观察到在低胆固醇含量下 DLPC 膜中的 GlpG 蛋白酶活性降低，这在 DMPC 中没有观察到。 虽然 GlpG 活性的变化已经是由于脂质环境中的最小差异造成的，可能使膜内蛋白水解的变构调节成为可能，但与胆固醇介导的脂质双层组织和相位行为没有总体相关性。需要考虑其他因素，例如胆固醇对膜弯曲刚度和曲率能量的影响。总之，α 螺旋膜蛋白（如 GlpG）的功能不仅取决于疏水匹配，还取决于其他膜特性、特异性脂质相互作用和环层的组成。