Danner M, Seckler R
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Protein Sci. 1993 Nov;2(11):1869-81. doi: 10.1002/pro.5560021109.
Temperature-sensitive folding (tsf) and global-tsf-suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off-pathway aggregation, the kinetics of refolding after dilution from acid-urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants. At low temperature (10 degrees C), subunit-folding rates, measured as an increase in fluorescence, were similar for wild-type and mutants. At 25 degrees C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf-mutant background, but had no effect in the wild-type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off-pathway reaction, as revealed by temperature down-shifts after varied times during refolding at high temperature (40 degrees C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate. In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up-shifts during refolding at 25 degrees C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit-folding intermediates and were also observable during thermal unfolding of the native protein.
温度敏感折叠（tsf）和全局tsf抑制（su）点突变在体内和体外均会影响三聚体、热稳定噬菌体P22尾刺内鼠李糖苷酶在高温下的折叠产率，但对天然折叠蛋白的功能和稳定性影响较小。为了阐明这些突变改变正确折叠与错误折叠聚集之间分配的机制，分析了从酸性尿素溶液稀释后的复性动力学以及折叠中间体的热稳定性。该研究包括五个不同严重程度的tsf突变、两个已知的su突变以及四个tsf/su双突变体。在低温（10℃）下，以荧光增加来衡量的亚基折叠速率，野生型和突变体相似。然而，在25℃时，tsf突变降低了亚基折叠速率。当存在于tsf突变背景中时，su突变增加了该速率，但在野生型背景中没有影响。相反，如在高温（40℃）复性过程中不同时间后温度下降所显示的，tsf突变加速了，而su突变阻碍了不可逆的错误折叠反应。动力学结果与tsf突变使关键亚基折叠中间体不稳定以及su突变使其稳定相一致。根据这一解释，如在25℃复性过程中温度上升所揭示的，tsf突变降低了，而su突变增加了折叠中间体的耐热性。稳定和不稳定作用在复性早期最为明显。然而，它们并不局限于亚基折叠中间体，在天然蛋白的热解折叠过程中也可观察到。
