Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle α-tropomyosin in an engineered chimeric protein
Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle α-tropomyosin in an engineered chimeric protein
复制标题
DOI：
10.1006/jmbi.2001.4982
复制
发表时间：
2001-09-28
影响因子：
5.6
通讯作者：
Hitchcock-DeGregori, SE
中科院分区：
文献类型：
--
作者：
Greenfield, NJ;Huang, YJ;Hitchcock-DeGregori, SE
原肌球蛋白是一种α-螺旋螺旋卷曲蛋白，它沿着肌动蛋白细丝的长度从头到尾排列，并调节其功能。在一种名为GlyTM1bZip的工程嵌合蛋白中，确定了一个功能重要的短247个残基的非肌肉原肌球蛋白N末端的溶液结构。GlyTM1bZip由大鼠短α-肌球蛋白的前19个残基和Gcn4亮氨酸拉链的最后18个残基组成。合成了编码GlyTM1bZip的基因，并在大肠杆菌中进行了表达。用AutoAssign程序对其三重共振谱进行了分析，并对其主链进行了归属。用AutoStructure分析了多维核Overhauser效应谱、X-滤波谱和(3)J(H-N-H-α)标量耦合。这是首次应用这一新程序来确定对称同源二聚体和以前未报道的结构的三维结构。GlyTM1bZip中的7-35残基形成卷曲的线圈，但两端都不是螺旋的。异核N-15-H-1核Overhaser效应数据表明，非螺旋N-末端残基是柔性的。GlyTM1bZip中螺旋线圈主链羰基的C-13‘化学位移表现出以前未曾报道过的周期性，其中来自七重复序列a和d位置的螺旋线圈界面残基的共振相对前场位移，而来自c位残基的共振相对下场位移。收集的异核单量子相干谱随温度的变化表明，在盘管界面上来自α螺旋主链和侧链的交叉峰随着T-M值的增加而加宽或移动，T-M值比圆二色谱测量的α螺旋损失低20度C，这表明存在折叠中间体。Ile14的侧链是结合相互作用所必需的残基，表现出多种构象。短原肌球蛋白N末端的构象灵活性可能是其结合特异性的重要因素。(C)2001年学术出版社。
Tropomyosin is an alpha -helical coiled-coil protein that aligns head-to-tail along tl e length of the actin filament and regulates its function. The solution structure of the functionally important N terminus of a short 247-residue non-muscle tropomyosin was determined in an engineered chimeric protein, GlyTM1bZip, consisting of the first 19 residues of rat short a-tropomyosin and the last 18 residues of the GCN4 leucine zipper. A gene encoding GlyTM1bZip was synthesized, cloned and expressed in Escherichia coli. Triple resonance NMR spectra were analyzed with the program AutoAssign to assign its backbone resonances. Multidimensional nuclear Overhauser effect spectra, X-filtered spectra and (3)J(H-N-H-alpha) scalar coupling were analyzed using AutoStructure. This is the first application of this new program to determine the three-dimensional structure, of a symmetric homodimer and a structure not previously reported. Residues 7-35 in GlyTM1bZip form a coiled coil, but neither end is helical. Heteronuclear N-15-H-1 nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible. The C-13' chemical shifts of the coiled-coil backbone carbonyl groups in GlyTM1bZip showed a previously unreported periodicity, where resonances arising from residues at the coiled-coil interface in a and d positions of the heptad repeat were displaced relatively upfield and those arising from residues in c positions were displaced relatively downfield. Heteronuclear single quantum coherence spectra, collected as a function of temperature, showed that cross-peaks arising from the alpha -helical backbone and side-chains at the coiled-coil interface broadened or shifted with T-M values similar to 20 degreesC lower than the loss of alpha -helix measured by circular dichroism, suggesting the presence of a folding intermediate. The side-chain of Ile14, a residue essential for binding interactions, exhibited multiple conformations. The conformational flexibility of the N termini of short tropomyosins may be important for their binding specificity. (C) 2001 Academic Press.