FOLDING STUDIES OF SINGLE TRP MUTANTS OF RNASE A

我们已经制造了一个核糖核酸酶A的突变体，它包含一个单一的 色氨酸残基(Trp92)。这个突变体已经知道了荧光 属性，这些属性可以探测与 热能展开。我们已经在PH值中诱导了190摄氏度的温度跃升 4.5用纳秒OPO产生10 mJ ns脉冲(1.5m)的Y92W溶液； 突变体中Trp残基(340 Nm)的荧光为 从T跳跃前的-500 ns到T跳跃后的30 ms的时间进行监测 T-跳跃。调整蛋白质溶液的静态温度 从250摄氏度到550摄氏度(通过熔化曲线)。数据支持这一点 色氨酸残基周围没有明显的扰动 时间刻度小于30毫秒。此外，一个显著的差异是 在提高温度之间的荧光强度 变性和盐酸铵变性也很明显，这表明 不同的变性机制。

We have made a mutant of Ribonuclease A which contains a single tryptophan residue (Trp92). This mutant has known fluorescent properties which can probe conformational changes associated with thermal unfolding. We have induced a 190C temperature jump in a pH 4.5 solution of Y92W using a 10 mJ ns pulse (1.5 ?m) from the ns OPO; the fluorescence from the Trp residue (at 340 nm) in the mutant is monitored at times from -500 ns before the T-jump to 30 ms after the T-jump. The static temperature of the protein solution was adjusted from 250C to 550C (through the melting curve). The data support that no significant perturbation surrounding the Trp residue is evident on timescales shorter than 30 ms. In addition, a significant difference in the increase of fluorescent intensity between temperature denaturation and GuHCl denaturation is also evident, suggesting differing mechanisms for denaturation.