CAREER: Structural Studies of a Highly Conserved Dynein Light Chain and its Role in Dynein Assembly and Cargo Recruitment

细胞质动力蛋白是一种马达蛋白复合物，在许多过程中是必不可少的，包括有丝分裂期间的染色体分离，囊泡货物的运动，细胞内细胞器的定位，以及卵母细胞发育中细胞极性的建立。本项目主要研究果蝇动力蛋白轻链LC 8在动力蛋白组装和货物识别中的作用。LC 8是一个高度保守的基因，果蝇和人类之间的同源性高达94%。动力蛋白中间链IC 74介导重链和轻链之间的相互作用，LC 8和IC 74的组装可能是形成功能复合物的关键步骤。为了阐明LC 8/IC 74识别的基础，将使用NMR光谱法确定复合物的结构。作为货物的一个例子，吞咽蛋白Swa是一种RNA结合蛋白，其在发育中的卵母细胞中建立细胞极性中至关重要。Swa与LC 8的相互作用已在果蝇中得到证实。在这种相互作用的结合位点的鉴定将由有限的蛋白水解和交联studies.Broader影响：LC 8及其与IC 74和Swa的相互作用的结构研究将是一个重大贡献，了解组装的动力蛋白电机复合物和其附件货物。这项工作将需要一些本科生作出实质性贡献。该项目还将涉及开发一个关于结构生物学和生物物理学当前主题的课程，这些主题已成为许多生物研究的核心。

Cytoplasmic dynein is a motor protein complex that is essential in a number of processes including chromosome segregation during mitosis, movement of vesicular cargo, positioning of intracellular organelles, and the establishment of cell polarity in oocyte development. This project focuses on characterizing the role of dynein light chain LC8 from Drosophila in dynein assembly and cargo recognition. LC8 is highly conserved, with 94% sequence identity between Drosophila and human. The dynein intermediate chain IC74 mediates the interaction between the heavy and light chains, and the assembly of LC8 and IC74 is likely to be a key step in formation of a functional complex. To elucidate the basis for LC8/IC74 recognition, the structure of the complex will be determined using NMR spectroscopy. The swallow protein, Swa, an example of cargo, is an RNA binding protein, which is essential in establishing cell polarity in developing oocytes. The interaction of Swa with LC8 has been demonstrated in Drosophila. Identification of binding sites in this interaction will be determined by limited proteolysis and cross-linking studies.Broader Impact: Structural studies of LC8 and its interactions with IC74 and Swa will be a significant contribution to understanding both the assembly of the dynein motor complex and its attachment to cargo. This work will entail substantive contributions from a number of undergraduates. The project will also involve development of a class on the current topics in structural biology and biophysics, which have become central to much of biological research.