A Novel Method of Determining the Functional Effects of a Minor Genetic Modification of a Protein.
A Novel Method of Determining the Functional Effects of a Minor Genetic Modification of a Protein.
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DOI：
10.3389/fcvm.2015.00035
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发表时间：
2015
影响因子：
3.6
通讯作者：
Borejdo J
中科院分区：
文献类型：
--
作者：
Nagwekar J;Duggal D;Midde K;Rich R;Liang J;Kazmierczak K;Huang W;Fudala R;Gryczynski I;Gryczynski Z;Szczesna-Cordary D;Borejdo J
肌肉的收缩是由肌球蛋白跨桥与肌动蛋白丝的ATP偶联循环相互作用引起的。当转基因蛋白表达水平低时，收缩的宏观参数，如最大张力、缩短速度或ATP酶活性，不太可能揭示野生型和突变（MUT）蛋白之间的差异。这是因为宏观测量是在含有数万亿肌动蛋白和肌球蛋白分子的整个器官上进行的。从这样一个大的集合体收集的信息的平均值必然会掩盖由一小部分MUT分子施加的任何差异。为了避免平均化问题，在分离的心室肌原纤维（MF）上进行测量，其中细丝稀疏地用荧光染料标记。我们从心室中分离出一个单一的MF，将其垂直定向（以便能够测量方向），并用荧光染料标记十万分之一的肌动蛋白单体。我们观察到的荧光从一个小的共聚焦体积含有大约三个肌动蛋白分子。在心室收缩期间，肌动蛋白不断地改变取向（即，刚性连接的荧光团的跃迁时刻随时间波动），因为它被肌球蛋白横桥重复地“踢”。这些波动的自相关函数（ACFs）对肌球蛋白的突变非常敏感。我们研究了人群研究显示的肌球蛋白调节轻链丙氨酸转苏氨酸（A13 T）突变对肥厚型心肌病的影响。这是一个恰当的例子，因为在转基因小鼠的心室中突变仅以10%表达。ACF是“标准”（Std）（随时间单调衰减）或“非标准”（NStd）（不规则衰减）。肌动蛋白的稀疏标记也允许测量肌动蛋白分子的空间分布。这种分布反映了肌动蛋白与肌球蛋白跨桥的相互作用，并且对肌球蛋白突变也非常敏感。结果表明，A13 T突变导致9%的ACF和9%的肌动蛋白空间分布为NStd，其余91%为Std，表明NStd性能由MUT肌球蛋白头执行，而Std性能由非MUT肌球蛋白头执行。我们的结论是，在这项研究中探索的方法是一个敏感的和有效的测试低患病率突变的肌节蛋白质的属性。
Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being “kicked” by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either “Standard” (Std) (decaying monotonically in time) or “Non-standard” (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.