Engberg Oskar, Mathath Anjana V, Döbel Viola, Frie Christian, Lemberg Marius K, Chakraborty Debashree, Huster Daniel
Institute for Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany.
Biophysical and Computational Chemistry Laboratory, Department of Chemistry, National Institute of Technology Karnataka, Mangalore, Karnataka, India.
Biophys J. 2024 Dec 3;123(23):4067-4081. doi: 10.1016/j.bpj.2024.10.019. Epub 2024 Nov 1.
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 H and P 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.
细胞膜呈现出种类繁多的脂质和膜蛋白，它们在性质和化学结构上存在差异。细胞将这些分子组织成不同的膜区室，这些区室具有各自特定的脂质分布和疏水厚度。如果膜蛋白与周围脂质之间出现疏水不匹配，可能会产生功能后果，如蛋白质活性降低。对于单通道跨膜蛋白、视紫红质以及短杆菌肽等小多肽，这一现象已得到广泛研究。在此，我们以大肠杆菌菱形膜内蛋白酶GlpG为模型，系统地探究膜厚度对GlpG活性的影响。我们使用了完全饱和的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。通过氢核磁共振和磷核磁共振光谱测定物理膜参数，并将其与相应宿主膜中的GlpG活性测量结果相关联。使用分子动力学模拟确定相应宿主膜中本体脂质和环形脂质的差异以及蛋白质结构的变化。我们的研究结果表明，GlpG可以影响DLPC/胆固醇膜中的膜厚度，但不能影响DMPC/胆固醇膜中的膜厚度。此外，我们观察到在低胆固醇含量的DLPC膜中GlpG蛋白酶活性降低，而在DMPC膜中未观察到这种情况。虽然脂质环境中最小的差异就可能导致GlpG活性发生变化，这可能使膜内蛋白水解受到变构调节，但与胆固醇介导的脂质双层组织和相行为并无总体相关性。还需要考虑其他因素，如胆固醇对膜弯曲刚度和曲率能的影响。总之，像GlpG这样的α - 螺旋膜蛋白的功能不仅依赖于疏水匹配，还依赖于其他膜性质、特定的脂质相互作用以及环形层的组成。
