KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

KRas4b 是一种膜结合调节蛋白，属于小 GTP 酶家族，起分子开关的作用，促进信号从活化的膜受体转导到控制细胞生长和增殖的细胞内途径。将 KRas4b 锁定在活性 GTP 状态的致癌突变是导致近 85% 的 Ras 驱动癌症的原因。了解 KRas4b 的膜结合状态对于设计针对致癌 KRas 驱动的信号通路的新治疗方法至关重要。广泛的研究表明，膜双层在 Ras 效应子相互作用中具有重要意义，阴离子脂质在决定蛋白质构象中起关键作用 KRas4b 与信号转导伴侣相互作用的首选拓扑结构一直是一个长期的问题。计算研究表明膜近端构象，而其他生物物理方法（如中子反射率）则提出了膜远端构象。为了解决这些差距，我们采用 FRET 测量来研究 KRas4b 的构象。使用完全翻译后修饰的 KRas4b，我们设计了一种基于 Nanodisc 的 FRET 测定来研究 KRas4b 膜相互作用。我们建议 KRas4b 相对于膜表面的扩展构象。FRET供体-受体距离的测量表明，带负电荷的膜表面较弱地有利于与膜表面更紧密的结合。我们的研究结果为阴离子脂质在确定 KRas4b 的动态构象中的作用提供了见解，并阐明了其拓扑结构在脂质头基上的主要构象。