Interactions of Dynein Light and Intermediate Chains

描述（由申请人提供）：本提案侧重于细胞质动力蛋白两个亚基的相互作用：LC8，一个10 kDa的轻链，和IC74，一个74 kDa的中间链。我们在黑腹果蝇中过表达了这些亚基，并通过亲和方法、有限蛋白水解和圆二色性表征了它们在体外的相互作用。我们的第一个目标是确定每个亚基上负责相互作用的残基。我们将结合位点定位在IC74上K130附近的片段上。我们将使用核磁共振来识别原子尺度上的相互作用，并为此目的设计和制备了IC74结构。结合残基的核磁共振鉴定涉及到在异核磁共振实验中化学位移、弛豫速率和线宽的局部变化的确定。我们的第二个目标是表征IC74与LC8结合后的构象变化。我们观察到，与LC8结合会导致IC74 n端结构域的圆二色光谱发生巨大变化。这些变化与IC74中预测的一个或两个线圈区更大的排序是一致的。由于螺旋基序的典型功能是形成多亚基蛋白复合物，因此这些区域的高排序会增加IC74与动力蛋白其他亚基结合的倾向，而LC8与IC74的结合显然是复合物组装的启动因子。我们将使用核磁共振技术来研究结合和自由IC74结构的动力学，并定位与增加顺序相关的片段。我们还将使用氢交换质谱法通过测量结合和游离之间溶剂可及性的变化来检测LC8结合对增加IC74结构和稳定性的影响。我们之前已经在单体和二聚体LC8的动力学研究中证明了这种多功能技术的有效性。在这项工作中开发的方法将对我们正在进行的细胞质动力蛋白组装和功能的结构和动力学基础的研究越来越重要。这些研究将很快包括LC14和动力蛋白的其他亚基、辅助复合体动力蛋白和动力蛋白货物。与一位动力蛋白生物学专家合作，我们计划在果蝇中检查我们的发现的功能相关性。

DESCRIPTION (provided by applicant): This proposal focuses on interactions of two subunits of cytoplasmic dynein: LC8, a 10 kDa light chain, and IC74, the 74 kDa intermediate chain. We have overexpressed these subunits from Drosophila melanogaster and characterized their interactions in vitro by affinity methods, limited proteolysis, and circular dichroism. Our first aim is to identify the residues on each subunit which are responsible for the interaction. We have localized the binding site to the segment in the vicinity of K130 on IC74. We will use NMR to identify interactions at the atomic scale, with constructs of IC74 designed and prepared for this purpose. Identification of the binding residues by NMR involves the determination of local changes in chemical shifts, relaxation rates and line widths in heteronuclear NMR experiments. Our second aim is to characterize the conformational changes in IC74 upon binding to LC8. We have observed that binding to LC8 causes a dramatic change in circular dichroism spectra of the N-terminal domain of IC74. These changes are consistent with greater ordering in one or both of the coiled-coil regions predicted to be in IC74. Since the typical function of the coiled-coil motif is to nucleate the formation of multi-subunit protein complexes, greater ordering in these regions would increase the propensity of IC74 to bind to other subunits of dynein, and LC8 binding to IC74 would be clearly implicated as an initiating factor in the assembly of the complex. We will use NMR techniques to study the dynamics of bound and free IC74 constructs, and locate the segments involved in the increased order. We will also use hydrogen exchange mass spectrometry to examine the effect of LC8 binding on increasing structure and stability of IC74 by measurements of changes in solvent accessibility between bound and free. We have previously demonstrated the efficacy of this versatile technique in a study of the dynamics of monomeric and dimeric LC8.The methods that will be developed in this work will be increasingly important in our ongoing investigation of the structural and dynamic basis for the assembly and function of cytoplasmic dynein. These investigations will shortly encompass LC14 and other subunits of dynein, the accessory complex dynactin, and dynein cargo. With a collaborator who is an expert in dynein biology, we plan to examine the functional relevance of our findings in Drosophila.