Kuhlman B, Boice J A, Fairman R, Raleigh D P
Department of Chemistry, State University of New York, Stony Brook 11794-3400, USA.
Biochemistry. 1998 Jan 27;37(4):1025-32. doi: 10.1021/bi972352x.
The N-terminal domain, residues 1-56, of the ribosomal protein L9 has been chemically synthesized. The isolated domain is monomeric as judged by analytical ultracentrifugation and concentration-dependent CD. Complete 1H chemical shift assignments were obtained using standard methods. 2D-NMR experiments show that the isolated domain adopts the same structure as seen in the full-length protein. It consists of a three-stranded antiparallel beta-sheet sandwiched between two helixes. Thermal and urea unfolding transitions are cooperative, and the unfolding curves generated from different experimental techniques, 1D-NMR, far-UV CD, near-UV CD, and fluorescence, are superimposable. These results suggest that the protein folds by a two-state mechanism. The thermal midpoint of folding is 77 +/- 2 degrees C at pD 8.0, and the domain has a delta G degree folding = 2.8 +/- 0.8 kcal/mol at 40 degrees C, pH 7.0. Near the thermal midpoint of the unfolding transition, the 1D-NMR peaks are significantly broadened, indicating that folding is occurring on the intermediate exchange time scale. The rate of folding was determined by fitting the NMR spectra to a two-state chemical exchange model. Similar folding rates were measured for Phe 5, located in the first beta-strand, and for Tyr 25, located in the short helix between strands two and three. The domain folds extremely rapidly with a folding rate constant of 2000 s-1 near the midpoint of the equilibrium thermal unfolding transition.
核糖体蛋白L9的N端结构域（第1至56位氨基酸残基）已通过化学方法合成。通过分析超速离心和浓度依赖性圆二色光谱判断，分离得到的结构域为单体形式。使用标准方法完成了1H化学位移归属。二维核磁共振实验表明，分离得到的结构域与全长蛋白具有相同的结构。它由夹在两个螺旋之间的三股反平行β折叠片层组成。热变性和尿素变性转变是协同的，并且由不同实验技术（一维核磁共振、远紫外圆二色光谱、近紫外圆二色光谱和荧光）产生的变性曲线是可叠加的。这些结果表明该蛋白通过两态机制折叠。在pD 8.0时，折叠的热中点为77±2℃，该结构域在40℃、pH 7.0时的ΔG°折叠值为2.8±0.8 kcal/mol。在变性转变的热中点附近，一维核磁共振峰显著变宽，表明折叠发生在中间交换时间尺度上。通过将核磁共振谱拟合到两态化学交换模型来确定折叠速率。对位于第一条β链中的苯丙氨酸5和位于第二条链与第三条链之间短螺旋中的酪氨酸25测量了相似的折叠速率。在平衡热变性转变的中点附近，该结构域以2000 s-1的折叠速率常数极快速地折叠。
