Interactions of Dynein Light and Intermediate Chains

动力蛋白轻链和中间链的相互作用

基本信息

批准号：
6505284
负责人：
ELISAR J BARBAR
金额：
$ 14.5万
依托单位：
OHIO UNIVERSITY ATHENS
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-04-01 至 2004-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6505284
关键词：
Drosophilidae SDS polyacrylamide gel electrophoresis binding sites circular dichroism crosslink dimer dynein ATPase enzyme structure fluorescence spectrometry intracellular transport mass spectrometry molecular assembly /self assembly nuclear magnetic resonance spectroscopy polymerase chain reaction protein binding protein protein interaction protein structure function proteolysis

项目摘要

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.

描述（由申请人提供）：该提案的重点是两个细胞质动力蛋白的亚基的相互作用：LC8，10 kDa轻链和74 kDa中间链IC74。我们已经从果蝇中过表达了这些亚基，并通过亲和力方法，有限的蛋白水解和圆形二科主义在体外表征了它们的相互作用。我们的第一个目的是确定每个亚基上负责相互作用的残留物。我们已经将结合位点定位于IC74 K130附近的片段。我们将使用NMR在原子量表上识别交互作用，并为此目的设计和准备IC74的结构。 NMR对结合残基的鉴定涉及确定异核NMR实验中化学位移，松弛率和线宽度的局部变化。我们的第二个目的是表征与LC8结合后IC74中的构象变化。我们已经观察到，与LC8的结合会导致IC74 N末端结构域的圆形二色谱谱发生急剧变化。这些变化与预计在IC74中的一个或两个盘绕线圈区域的排序相一致。由于盘绕圈基序的典型功能是成核多生基蛋白络合物的形成，因此在这些区域中，更大的排序将增加IC74与Dynein的其他亚基结合的倾向，而LC8与IC74结合的倾向将与IC74结合，将与IC74结合，将其与综合体组装中的启动因子有关。我们将使用NMR技术研究结合和游离IC74构建体的动力学，并找到涉及增加顺序的段。我们还将使用氢交换质谱法检查LC8结合对IC74结构和稳定性的影响，通过测量结合和自由之间的溶剂可及性的变化。我们以前已经证明了这种多功能技术在对单体和二聚体LC8的动力学研究中的功效。在我们正在进行的对细胞质动力蛋白的组装和动态基础的研究中，这项工作中将开发的方法将变得越来越重要。这些调查将很快涵盖LC14和Dynein的其他亚基，附件复合型Dynactin和Dynein货物。与Dynein Biology专家的合作者一起，我们计划研究我们在果蝇中发现的功能相关性。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Characterization of the cargo attachment complex of cytoplasmic dynein using NMR and mass spectrometry.

使用 NMR 和质谱法表征细胞质动力蛋白的货物附着复合物。

DOI：
10.1016/s0076-6879(04)80011-9
发表时间：
2004
期刊：
Methods in enzymology.
影响因子：
0
作者：
Barbar,Elisar;Hare,Michael
通讯作者：
Hare,Michael

The solution structure of the pH-induced monomer of dynein light-chain LC8 from Drosophila.

pH 诱导的果蝇动力蛋白轻链 LC8 单体的溶液结构。

DOI：
10.1110/ps.03462204
发表时间：
2004
期刊：
Protein science : a publication of the Protein Society
影响因子：
0
作者：
Makokha,Moses;Huang,YuanpengJanet;Montelione,Gaetano;Edison,ArthurS;Barbar,Elisar
通讯作者：
Barbar,Elisar

Interactions of LC8 with N-terminal segments of the intermediate chain of cytoplasmic dynein.

LC8 与细胞质动力蛋白中间链 N 端片段的相互作用。