Musco G, Tziatzios C, Schuck P, Pastore A
EMBL, Heidelberg, Germany.
Biochemistry. 1995 Jan 17;34(2):553-61. doi: 10.1021/bi00002a021.
Titin, also known as connectin, is a giant modular protein specifically found in vertebrate striated muscle. Since the huge size of titin does not allow a direct structure determination, we have started a long-term project to characterize the protein by cutting it into smaller domains or structural units. The major part of the titin sequence is assembled by modules approximately 100 amino acids long that belong to two major protein superfamilies. Most of these modules are linked together by stretches of variable length with unique sequence. No direct structural characterization has been achieved so far for any of these linkers. We present here a study of a stretch located in the titin N-terminus and part of a linker between two modules. Our attention was drawn toward this region because it shows 100% probability to form a coiled coil when analyzed by a prediction program. A synthetic 38 amino acid peptide spanning such a sequence was studied in aqueous solution by circular dichroism, nuclear magnetic resonance, and analytical ultracentrifugation at various pH, salt, and peptide concentrations. Under all conditions, it shows a strong tendency to form alpha-helical structures. In the presence of salt, this conformation is associated with the formation of helical bundles below pH 5. Above pH 5, any aggregate breaks, and the titin peptide is a monomeric helix in equilibrium with its random coil conformation. We discuss the factors which stabilize the helical conformation and the possible role of this stretch in vivo.
肌联蛋白，也称为连接蛋白，是一种在脊椎动物横纹肌中特有的巨大模块化蛋白质。由于肌联蛋白的巨大尺寸不允许直接确定其结构，我们启动了一个长期项目，通过将其切割成较小的结构域或结构单元来对该蛋白质进行表征。肌联蛋白序列的主要部分由大约100个氨基酸长的模块组装而成，这些模块属于两个主要的蛋白质超家族。这些模块中的大多数通过具有独特序列的不同长度的片段连接在一起。到目前为止，尚未对这些连接子中的任何一个进行直接的结构表征。我们在此展示了一项针对位于肌联蛋白N端以及两个模块之间的一个连接子部分的片段的研究。我们的注意力被吸引到这个区域，因为通过预测程序分析时，它显示出100%形成卷曲螺旋的可能性。通过圆二色性、核磁共振以及在不同pH值、盐浓度和肽浓度下的分析超速离心，对跨越该序列的一条38个氨基酸的合成肽在水溶液中进行了研究。在所有条件下，它都表现出形成α螺旋结构的强烈倾向。在有盐存在的情况下，在pH值低于5时，这种构象与螺旋束的形成相关。在pH值高于5时，任何聚集体都会分解，并且肌联蛋白肽是一种单体螺旋，与其无规卷曲构象处于平衡状态。我们讨论了稳定螺旋构象的因素以及该片段在体内可能发挥的作用。
