What Region of Tropomyosin Interacts with the N-terminal Half of Troponin T?
What Region of Tropomyosin Interacts with the N-terminal Half of Troponin T?
复制标题
原肌球蛋白的哪个区域与肌钙蛋白 T 的 N 端一半相互作用？
DOI：
10.1016/j.bpj.2009.12.1901
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发表时间：
2010
影响因子：
3.4
通讯作者：
S. Lehrer
中科院分区：
文献类型：
--
作者：
A. Mudalige;S. Lehrer
原肌球蛋白（Tropomyosin，Tm）是一种α-螺旋卷曲螺旋蛋白，是肌肉收缩的关键调节蛋白。很少有人知道Tm的动态肌肉调节的作用，更具体地说，在三个国家的细丝的动态。在这项工作中，四个不同区域的Tm的灵活性测定与饱和转移电子顺磁共振（STEPR）。双功能标记的使用允许我们将探针固定并防止标记相对于蛋白质表面的运动。旋转相关时间的双功能自旋标记的Tm为40 ns相比，25 ns的常规单齿自旋标记。将自旋标记附接至通过半胱氨酸诱变获得的卷曲螺旋的第1、第4和第4位置。将双功能标记的Tm双突变体重组为“鬼肌纤维”，从中去除肌球蛋白丝和内在调节蛋白（原肌球蛋白，肌钙蛋白）。肌丝用肌球蛋白S1修饰。我们发现，有一个梯度的灵活性的Tm沿着其长度在肌纤维与C-末端突变体A268 C/E272 C是较低的移动的（两倍），与其余的突变体相比。肌钙蛋白的引入降低了四种突变体的柔性，特别是两种中间区域突变体H153 C/D157 C和G188 C/E192 C，分别降低了25%和30%。钙的添加确实显示C末端突变体A268 C/E272 C的柔性降低20%。另外两个突变体的加钙效果和S1对4个突变体的加钙效果不显著。因此，虽然有一个梯度的灵活性，在Tm，其动力学不改变不同状态的细丝激活。1820-Pos比较的构象稳定性的条纹α-原肌球蛋白适应不同的温度制度大卫H。希利，迈克尔·海莉，塔蒂阿娜·切瓦尔迪娜.加拿大圣约翰纪念大学。采用圆二色谱（CD）和差示扫描量热法比较了温血和冷血来源的α-横纹原肌球蛋白的构象稳定性。来自兔和鲨鱼的α-原肌球蛋白共享95%的序列同一性。核心中有三个替换：残基Thr 179 Ala、Ser 190 Cys和Ser 211 Ala。在低温（pH 7）下，两种原肌球蛋白在222 nm处的摩尔椭圆率相当，而鲨鱼蛋白在222 nm处的摩尔椭圆率相当。
Tropomyosin (Tm), an alpha-helical coiled-coil protein, is a key regulatory protein in muscle contraction. Little is known about the role of Tm dynamics in muscle regulation and more specifically dynamics in the three states of the thin filament. In this work, the flexibility of four different regions of Tm was determined with Saturation Transfer Electron Paramagnetic Resonance (STEPR). The use of bi-functional labels allowed us to immobilize the probe and prevent the motion of the label with respect to protein surface. The rotational correlation time of the bi-functional spin label on Tm was 40 ns compared to 25 ns for a conventional mono-dentate spin label. The spin label was attached to i, iþ4 positions of the coiled-coil, obtained by cysteine mutagenesis. The bi-functionally labeled Tm di-mutants were reconstituted into ‘‘ghost muscle fibers’’from which the myosin filaments and intrinsic regulatory proteins (tropomyosin, troponin) were removed. The filaments were reconsituted with Tn and decorated with myosin S1. We found that there is a gradient of flexibility of Tm along its length in the muscle fiber with the C-terminus mutant A268C/E272C being less mobile (two-fold), as compared to the rest of the mutants. Introduction of troponin decreases the flexibility of the four mutants, specifically the two mid-region mutants H153C/D157C and G188C/E192C by 25% and 30% respectively. The addition of calcium did show a decrease in the flexibility of the C terminus mutant A268C/E272C by 20%. The effect of calcium addition on the two other mutants and the effect of addition of S1 on the four mutants were not significant. Thus, although there is a gradient of flexibility in Tm, its dynamics does not change within different states of thin filament activation.1820-Pos Comparison of the Conformational Stabilities of Striated Alpha-Tropomyosins Adapted to Different Temperature Regimes David H. Heeley, Michael Hayley, Tatiana Chevaldina. Memorial Univeristy, St. John’s, NL, Canada. The conformational stabilities of alpha-striated tropomyosins from warm and cold blooded sources have been compared using circular dichroism (CD) and differential scanning calorimetry. Alpha-tropomyosins from rabbit and shark share 95% sequence identity. There are three replacements in the core: residues Thr179Ala, Ser190Cys and Ser211Ala. At low temperature (pH 7) the two tropomyosins are equivalent in molar ellipticity at 222nm, however, shark