Determinants of membrane protein stability and folding investigated by solid state NMR

通过固态核磁共振研究膜蛋白稳定性和折叠的决定因素

• 批准号: RGPIN-2020-04489
• 负责人: Ladizhansky, Vladimir
• 金额: $ 4.23万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739988
• 关键词: Determinants; membrane; protein; stability; folding

Membrane proteins play many important roles in biology of the cell, from regulation of membrane transport to signal transduction. These essential biomolecules have evolved to perform their function in cell membranes. The overarching long-term goal of our research program is to build an understanding of the interplay between membrane protein structure, dynamics and function and the cell membrane environment. One of the essential questions is how membrane proteins achieve their native functional structures. Folding of membrane proteins (a process by which a polypeptide chain adopts its native structure) is substantially more complex than that of their soluble counterparts, as in addition to intraprotein interactions, the presence of amphipathic lipid bilayer creates physical boundaries that limit the number of conformations accessible to membrane proteins. Furthermore, there is a growing body of evidence that specific and non-specific protein-lipid interactions, oligomerization, as well as interactions with ligands and cofactors play major roles in defining folding pathways and in stabilizing membrane protein structures. Understanding these interactions is the key to understanding of one of the central questions in membrane protein folding - how protein structure is established within membranes. We have previously focused on the development of solid-state nuclear magnetic resonance (SSNMR) methodologies that can provide the atomic level detail on membrane protein conformations in lipids and in cell membranes. We used these methods to characterize structure and dynamics of a seven-transmembrane helical retinal-binding photoreceptor Anabaena Sensory Rhodopsin (ASR). Our previous studies strongly suggest that the oligomeric structure of ASR is stabilized by interactions with tightly bound lipids, and these interactions modulate its folding landscape. Using this well-characterized model protein, we will study the relationship between ASR interactions and its folding. Our aims are (i) to determine the structural roles of lipids and of protein-protein interactions in the stability of ASR; (ii) to determine structures of the thermally induced unfolding intermediates and of the misfolded state, and to delineate the roles of cofactors and lipids in modulating the unfolding pathways.  Unfolding pathways will be detected by SSNMR and complementary techniques (e.g., Electron Paramagnetic Resonance), using either methods established by us earlier, or by trapping intermediate states by flash freezing, and by detecting chemical shift perturbations and key interatomic distances as structural reporters. By investigating structures and revealing stabilizing interactions of partially unfolded and misfolded states, we will determine the key factors governing proper folding of membrane proteins, and gain more detailed understanding of the membrane protein folding mechanisms.

膜蛋白在细胞生物学中扮演着许多重要的角色，从膜运输的调节到信号转导。这些基本的生物分子已经进化成在细胞膜上发挥它们的功能。我们研究计划的总体长期目标是了解膜蛋白结构、动力学和功能与细胞膜环境之间的相互作用。其中一个基本问题是膜蛋白如何实现其天然的功能结构。膜蛋白的折叠(多肽链采用其天然结构的过程)比其可溶性蛋白的折叠要复杂得多，因为除了蛋白质内部的相互作用外，两亲性脂质双层的存在创造了物理边界，限制了膜蛋白可获得的构象数量。此外，越来越多的证据表明，特定和非特定的蛋白质-脂质相互作用、齐聚作用以及与配体和辅因子的相互作用在确定折叠途径和稳定膜蛋白结构方面发挥着重要作用。了解这些相互作用是理解膜蛋白质折叠的核心问题之一的关键--蛋白质结构是如何在膜内建立的。我们之前一直专注于固体核磁共振(SS核磁共振)方法的发展，这种方法可以提供脂类和细胞膜中膜蛋白构象的原子水平细节。我们使用这些方法来表征七跨膜螺旋视网膜结合光感受器鱼腥藻感觉视紫红质(ASR)的结构和动力学。我们以前的研究强烈表明，ASR的低聚结构是通过与紧密结合的脂类相互作用来稳定的，这些相互作用调节了它的折叠景观。利用这一特性良好的模型蛋白，我们将研究ASR相互作用与其折叠之间的关系。我们的目标是(I)确定脂类和蛋白质-蛋白质相互作用在ASR稳定性中的结构作用；(Ii)确定热诱导展开中间产物和错误折叠状态的结构，并描述辅因子和脂类在调节展开途径中的作用。我们将使用我们先前建立的方法或通过闪速冷冻捕获中间态，并通过检测化学位移扰动和关键的原子间距离作为结构记者，通过SS核磁共振和辅助技术(例如电子顺磁共振)来检测展开途径。通过研究结构和揭示部分未折叠和错误折叠状态的稳定相互作用，我们将确定控制膜蛋白正确折叠的关键因素，并对膜蛋白折叠机制有更详细的了解。