Greenfield N J, Huang Y J, Palm T, Swapna G V, Monleon D, Montelione G T, Hitchcock-DeGregori S E
Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
J Mol Biol. 2001 Sep 28;312(4):833-47. doi: 10.1006/jmbi.2001.4982.
Tropomyosin is an alpha-helical coiled-coil protein that aligns head-to-tail along the 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 alpha-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 (15)N-(1)H nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible. The (13)C' 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 approximately 20 degrees C 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.
原肌球蛋白是一种α-螺旋卷曲螺旋蛋白，它沿肌动蛋白丝的长度首尾对齐排列，并调节其功能。在一种工程化嵌合蛋白GlyTM1bZip中确定了一种由247个残基组成的短非肌肉原肌球蛋白功能重要的N端的溶液结构，该嵌合蛋白由大鼠短α-原肌球蛋白的前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，将其克隆并在大肠杆菌中表达。用AutoAssign程序分析三重共振NMR谱以归属其主链共振。使用AutoStructure分析多维核Overhauser效应谱、X-滤波谱和(3)J(H(N)-H(α))标量耦合。这是该新程序首次用于确定对称同二聚体的三维结构以及一个此前未报道的结构。GlyTM1bZip中的7至35位残基形成一个卷曲螺旋，但两端均无螺旋结构。异核(15)N-(1)H核Overhauser效应数据表明，非螺旋的N端残基具有柔性。GlyTM1bZip中卷曲螺旋主链羰基的(13)C'化学位移呈现出一种此前未报道的周期性，其中七肽重复序列a和d位置的卷曲螺旋界面处残基产生的共振相对向高场位移，而c位置残基产生的共振相对向低场位移。作为温度函数收集的异核单量子相干谱表明，卷曲螺旋界面处α-螺旋主链和侧链产生的交叉峰随着T(M)值变宽或位移，其T(M)值比通过圆二色性测量的α-螺旋丧失温度低约20℃，这表明存在折叠中间体。Ile14的侧链（一种对于结合相互作用至关重要的残基）呈现出多种构象。短原肌球蛋白N端的构象柔性可能对其结合特异性很重要。
