Saraswat L D, Lowey S
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
J Biol Chem. 1991 Oct 15;266(29):19777-85.
Site-directed mutagenesis has been used to insert cysteine residues at specific locations in the myosin light chain 2 (LC2) sequence. The aim was to modify these cysteines with one or more spectroscopic probes and to reconstitute myosin with labeled light chains for structural studies. Native LC2 has two endogenous cysteine residues at positions 126 and 155; a third sulfhydryl was added by replacing either Pro2, Ser73, or Pro94 with cysteine. By oxidizing the endogenous cysteines to an intramolecular disulfide bond (Katoh, T., and Lowey, S., (1989) J. Cell Biol. 109, 1549), it was expected that the new cysteine could be selectively labeled with a fluorescent probe. This proved more difficult to accomplish than anticipated due to the formation of secondary disulfide bonds between the newly engineered cysteines and the native ones. Nevertheless, the unpaired cysteines were labeled with 5-(iodoacetamido)fluorescein, and singly labeled species were purified by ion-exchange chromatography. Chymotryptic digestion of the light chains, followed by high performance liquid chromatography separation of the peptides, led to the identification of the fluorescein-labeled cysteines. After light chain exchange into myosin, the position of the thiols was mapped by antifluorescyl antibodies in the electron microscope. Rotary-shadowed images showed the antibody bound at the head/rod junction of myosin for all the mutants. These mapping studies, together with the finding that widely separated cysteines can form multiple disulfide bonds, support a model for LC2 as a flexible, globular molecule that resembles other Ca/Mg-binding proteins in tertiary structure.
定点诱变已被用于在肌球蛋白轻链2（LC2）序列的特定位置插入半胱氨酸残基。目的是用一种或多种光谱探针修饰这些半胱氨酸，并用标记的轻链重构肌球蛋白以进行结构研究。天然LC2在126位和155位有两个内源性半胱氨酸残基；通过用半胱氨酸取代Pro2、Ser73或Pro94添加了第三个巯基。通过将内源性半胱氨酸氧化成分子内二硫键（加藤，T.，和洛维，S.，（1989年）《细胞生物学杂志》109卷，1549页），预计新的半胱氨酸可以用荧光探针选择性标记。由于新设计的半胱氨酸与天然半胱氨酸之间形成了二级二硫键，这一过程比预期的更难完成。尽管如此，未配对的半胱氨酸用5-（碘乙酰胺基）荧光素标记，单标记的物种通过离子交换色谱法纯化。轻链经胰凝乳蛋白酶消化，随后通过高效液相色谱法分离肽段，从而鉴定出荧光素标记的半胱氨酸。将轻链交换到肌球蛋白中后，通过电子显微镜中的抗荧光酰基抗体定位硫醇的位置。旋转阴影图像显示，所有突变体的抗体都结合在肌球蛋白的头部/杆部连接处。这些定位研究，以及广泛分离的半胱氨酸可以形成多个二硫键的发现，支持了LC2是一种柔性球状分子的模型，其三级结构类似于其他钙/镁结合蛋白。
