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

项目名称：Beclin 1是一种进化上保守的关键自噬蛋白，其构象灵活性自噬是一种关键的细胞存活途径，由于不需要的、受损的或有害的细胞成分的降解而负责营养循环。 自噬在几个关键的生物过程中起着重要的作用，包括胚胎发育，组织分化，细胞生长，以及生存的环境压力和内部应激源，如受损的细胞器，突变蛋白，自由基和细胞内病原体。 Beclin 1是自噬所必需的蛋白质，直接参与了这些过程。 这项研究将侧重于了解Beclin 1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护中的关键作用。 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了解生物大分子结构和功能，在实验室外产生重大影响。 在该项目期间，40名高中科学教师将参加暑期专业发展课程，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，在这个项目中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;并将有一个更广泛的影响远远超出了赠款资助期。Beclin 1似乎是一个蛋白质相互作用中心的自噬，因为它结合到20多种不同的细胞蛋白质。 该项目的研究目标是更好地了解Beclin 1如何通过建立其结构和构象灵活性并识别功能关键的残基来与如此多的不同伙伴相互作用。 圆二色性和核磁共振（NMR）光谱将用于评估二级结构含量、主链柔性和动态构象变化。 高分辨率结构将通过X射线晶体学或NMR确定。小角X射线散射将用于获得低分辨率的结构信息，如大小，低聚和分子包膜。 从这些方法的组合信息将被用来推导出一个全长Beclin 1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，并通过细胞测定来评估突变对细胞自噬水平和所选关键自噬蛋白结合的影响。 最后，但并非最不重要的是，这项研究将提供大量的信息了解不多的内在无序区域，并帮助建立调查这些地区所必需的方法。

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.