Brandmier Katrina, Glover Kerney Jebrell
Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania, USA.
bioRxiv. 2025 Jul 12:2025.07.10.664188. doi: 10.1101/2025.07.10.664188.
Caveolin-1 (Cav1) is an integral membrane protein essential for the formation of caveolae, plasma microdomains implicated in signal transduction and mechanoprotection. Cav1 is comprised of three major alpha helices, but the topology these helices adopt remains unclear. Proline 110 is located between helix 1 and helix 2, and is hypothesized to enable Cav1 to adopt an intramembrane turn crucial for the cytosolic topology of Cav1. To assess the structural role of Proline 110, we utilized Förster resonance energy transfer (FRET) between native tryptophan (W128) and site-specifically labeled dansyl fluorophores to monitor conformational changes induced by the mutation of Proline 110 to Alanine (P110A). Static light scattering confirmed that all FRET constructs behaved monomerically, ensuring intramolecular energy transfer measurements. Our results show a significant decrease in FRET efficiency upon the P110A mutation consistent with a large conformational change. These findings support the critical role of P110 in maintaining the native topology of Cav1 and highlights the structural sensitivity of the intramembrane turn.
小窝蛋白-1（Cav1）是一种整合膜蛋白，对于小窝（参与信号转导和机械保护的质膜微区）的形成至关重要。Cav1由三个主要的α螺旋组成，但其采用的拓扑结构仍不清楚。脯氨酸110位于螺旋1和螺旋2之间，据推测它能使Cav1形成对其胞质拓扑结构至关重要的膜内转角。为了评估脯氨酸110的结构作用，我们利用天然色氨酸（W128）与位点特异性标记的丹磺酰荧光团之间的荧光共振能量转移（FRET）来监测脯氨酸110突变为丙氨酸（P110A）所诱导的构象变化。静态光散射证实所有FRET构建体均以单体形式存在，确保了分子内能量转移测量。我们的结果表明，P110A突变后FRET效率显著降低，这与大的构象变化一致。这些发现支持了P110在维持Cav1天然拓扑结构中的关键作用，并突出了膜内转角的结构敏感性。
