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的两个亚基的相互作用：LC 8（10 kDa轻链）和IC 74（74 kDa中间链）。我们已经过表达这些亚基从果蝇和其特点是在体外的相互作用，亲和方法，有限的蛋白水解，和圆二色性。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。我们已经将结合位点定位于IC 74上K130附近的片段。我们将使用NMR来识别原子尺度上的相互作用，并为此目的设计和制备IC 74的结构。通过NMR鉴定结合残基涉及在异相NMR实验中确定化学位移、弛豫速率和线宽的局部变化。我们的第二个目标是表征IC 74与LC 8结合后的构象变化。我们已经观察到，结合到LC 8引起的IC 74的N-末端结构域的圆二色光谱的显着变化。这些变化与预测在IC 74中的一个或两个卷曲螺旋区域中的更大排序一致。由于卷曲螺旋基序的典型功能是使多亚基蛋白质复合物的形成成核，因此这些区域中的更大有序性将增加IC 74与动力蛋白的其他亚基结合的倾向，并且LC 8与IC 74的结合将明确地暗示为复合物组装中的起始因子。我们将使用NMR技术来研究结合和游离IC 74构建体的动力学，并定位参与增加顺序的片段。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查LC 8结合对增加IC 74的结构和稳定性的影响。我们已经在单体和二聚体LC 8的动力学研究中证明了这种多功能技术的有效性。在这项工作中将发展的方法将在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中越来越重要。这些研究将很快涵盖LC 14和其他亚基的动力蛋白，辅助复合体动力蛋白，和动力蛋白货物。我们计划与动力蛋白生物学专家合作，研究我们在果蝇中发现的功能相关性。

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.