Investigating EnB1-Induced Membrane Remodeling via Contrast-Variation Neutron Scattering

Cellular membranes exhibit complex three-dimensional architectures essential for numerous biological processes. Membrane-binding proteins contribute to shaping these structures by inducing curvature and remodeling lipid bilayers into tubules and vesicles. Among them, proteins from the Bin/Amphiphysin/Rvs167 (BAR) domain superfamily are key players. These peripheral dimers possess a conserved coiled-coil domain that forms a banana-shaped BAR domain capable of sensing and generating curvature. N-BAR proteins, a subclass, also contain N-terminal amphipathic helices that insert into membranes to enhance curvature. Endophilin B1 (EnB1), an N-BAR protein, participates in oncogenesis-related pathways, including autophagy and apoptosis. Its amphipathic regions mediate membrane association and tubule formation. Cryo-EM has shown EnB1 decorates lipid vesicles and binds nanodiscs as dimers, but these structures do not fully resolve lipid bilayer rearrangements. To address this, we propose using contrast variation small-angle neutron scattering (SANS), which enables selective visualization of sample components by modulating the D₂O/H₂O ratio. Unlike X-rays, neutrons scatter based on nuclear scattering length density, allowing specific masking of components such as protein, lipid tails, or headgroups. This approach will enable detailed analysis of lipid bilayer remodeling induced by EnB1.

细胞膜具有复杂的三维结构，这对众多生物学过程至关重要。膜结合蛋白可通过诱导膜曲率、将脂质双分子层重塑为小管与囊泡，参与塑造这些膜结构。其中，Bin/Amphiphysin/Rvs167（BAR）结构域超家族蛋白是关键参与者。这类外周二聚体蛋白拥有保守的卷曲螺旋结构域，可形成香蕉形的BAR结构域，能够感知并产生膜曲率。作为该超家族的亚类，N-BAR蛋白还含有N端两亲性螺旋，可插入膜中以增强膜曲率。Endophilin B1（EnB1）作为一类N-BAR蛋白，参与包括自噬与凋亡在内的肿瘤发生相关通路，其两亲性区域介导膜结合与小管形成。冷冻电镜（Cryo-EM）研究显示，EnB1以二聚体形式装饰脂质囊泡并结合纳米圆盘，但这些结构未能完全解析脂质双分子层的重排过程。为解决这一问题，本研究拟采用对比度变化小角中子散射（SANS）技术，该技术可通过调节重水（D₂O）与普通水（H₂O）的比例，实现对样品组分的选择性可视化。与X射线不同，中子的散射基于核散射长度密度，因此可特异性遮蔽蛋白质、脂质尾链或头部基团等组分。该技术将实现对EnB1诱导的脂质双分子层重塑过程的精细化分析。