Conformational Flexibility in Beclin 1, an Evolutionarily-conserved, Key Autophagy Protein

Beclin 1（一种进化保守的关键自噬蛋白）的构象灵活性

• 批准号: 1413525
• 负责人: Sangita Sinha
• 金额: $ 89.08万
• 依托单位: North Dakota State University Fargo
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2020-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1413525&HistoricalAwards=false
• 关键词: Conformational; Flexibility; Beclin; Evolutionarily; conserved

Project title: Conformational flexibility in Beclin 1, an evolutionarily-conserved, key autophagy proteinAutophagy is a critical cell survival pathway responsible for nutrient recycling due to degradation of unwanted, damaged or harmful cellular components. Autophagy plays an important role in several key organismal processes including embryonic development, tissue differentiation, cell-growth, as well as surviving environmental stress and internal stressors like damaged organelles, mutant proteins, free radicals and intracellular pathogens. Beclin1, a protein essential for autophagy, has been directly implicated in these processes. This research will focus on understanding the atomic-resolution structure and flexibility of Beclin1 to provide a better understanding of its critical role in autophagy and cellular maintenance. This project will provide graduate and undergraduate students in North Dakota an unprecedented opportunity to train in classical and cutting edge methods of biophysical and biochemical research, and also have a substantial impact outside the laboratory by educating high-school teachers about biomacromolecular structure and function. Over the period of this project, 40 high-school science teachers will participate in a summer professional development course to help them instruct their students in understanding the structures of biomacromolecules such as proteins and DNA. Thus, the educational activities outlined in this project are designed to educate and mentor students at all levels: high school, undergraduate and graduate; and will have a broader impact far beyond the grant funding period.Beclin1 appears to be a protein interaction hub for autophagy as it binds to over twenty diverse cellular proteins. The research goal of this project is to better understand how Beclin 1 interacts with so many different partners by establishing its structure and conformational flexibility and identifying residues that are key for function. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy will be used to assess secondary structure content, backbone flexibility and dynamic conformational changes. High-resolution structures will be determined by X-ray crystallography or NMR. Small-angle X-ray scattering will be used to obtain low-resolution structural information such as size, oligomerization and molecular envelopes. Combined information from these methods will be used to derive a pseudo-atomic model of full-length Beclin1. The role of motifs and conserved residues in binding will be examined using isothermal titration calorimetry to quantify the thermodynamics of interaction and by cellular assays to assess the impact of mutations on cellular autophagy levels and binding of selected key autophagy proteins. Last, but not least, this research will provide substantial information about poorly understood intrinsically disordered regions and help establish methodology essential for investigating such regions.

项目名称：Beclin 1的构象灵活性，Beclin 1是一种进化保守的关键自噬蛋白自噬是一个关键的细胞生存途径，负责由于降解不需要的、受损的或有害的细胞成分而进行营养循环。自噬在包括胚胎发育、组织分化、细胞生长、生存环境压力和内源（如受损的细胞器、突变蛋白、自由基和细胞内病原体）在内的几个关键生物过程中起着重要作用。Beclin1是一种自噬所必需的蛋白质，直接参与了这些过程。本研究将重点了解Beclin1的原子分辨率结构和灵活性，以更好地了解其在自噬和细胞维持中的关键作用。该项目将为北达科他州的研究生和本科生提供一个前所未有的机会，在生物物理和生物化学研究的经典和前沿方法方面进行培训，并通过教育高中教师有关生物大分子结构和功能的知识，在实验室之外产生重大影响。在这个项目期间，40名高中科学教师将参加一个夏季专业发展课程，帮助他们指导学生了解蛋白质和DNA等生物大分子的结构。因此，本项目概述的教育活动旨在教育和指导各级学生：高中、本科和研究生；并将产生更广泛的影响，远远超出赠款资助期。Beclin1似乎是自噬的蛋白质相互作用中心，因为它与20多种不同的细胞蛋白质结合。该项目的研究目标是通过建立Beclin 1的结构和构象灵活性，以及识别对功能至关重要的残基，更好地了解Beclin 1如何与如此多不同的伙伴相互作用。圆二色性和核磁共振（NMR）光谱将用于评估二级结构含量、主链柔韧性和动态构象变化。高分辨率结构将通过x射线晶体学或核磁共振来确定。小角度x射线散射将用于获得低分辨率的结构信息，如尺寸，寡聚化和分子包膜。这些方法的综合信息将用于推导全长Beclin1的伪原子模型。基序和保守残基在结合中的作用将使用等温滴定量热法来量化相互作用的热力学，并通过细胞分析来评估突变对细胞自噬水平和选定的关键自噬蛋白结合的影响。最后，但并非最不重要的是，这项研究将提供关于缺乏理解的内在无序区域的大量信息，并帮助建立调查这些区域所必需的方法。