Hamman B D, Oleinikov A V, Jokhadze G G, Traut R R, Jameson D M
Department of Biochemistry and Biophysics, University of Hawaii, Honolulu 96822, USA.
Biochemistry. 1996 Dec 24;35(51):16672-9. doi: 10.1021/bi9615001.
Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.
利用荧光方法研究了24 kDa二聚体大肠杆菌核糖体蛋白L7/L12的动态特性。采用寡核苷酸定点诱变技术，在肽链的特定位置，即C端和N端结构域引入半胱氨酸残基，并将各种巯基反应性荧光探针（碘乙酰胺荧光素、IAEDANS、芘甲基碘乙酸酯）连接到这些残基上。除了全长蛋白外，还研究了一种缺失铰链的变体以及对应于C端片段和N端片段的变体。进行了稳态和时间分辨荧光测量，结果表明L7/L12不是一个刚性分子。具体而言，两个C端结构域相对于彼此以及相对于二聚体N端结构域可自由移动。然而，去除铰链区会显著降低C端结构域的流动性。数据还表明，由荧光探针监测的旋转弛豫时间取决于探针的激发态寿命。这一观察结果被解释为表明L7/L12分子中存在运动层次结构，除了蛋白质的整体翻滚外，还包括C端结构域和二聚体N端结构域的轻快运动。连接到N端结构域的探针表现出与二聚体N端片段分子量一致的整体旋转弛豫时间。然而，用核糖体核心重构标记的L7/L12后，与二聚体N端结构域相关的运动大大减少，而C端结构域的轻快运动几乎不变。
