Lin L N, Brandts J F
Department of Chemistry, University of Massachusetts, Amherst 01003.
Biochemistry. 1988 Dec 13;27(25):9037-42. doi: 10.1021/bi00425a023.
In an effort to determine structural properties of the nativelike intermediate (i.e., IN) which forms during the refolding of RNase A, refolding samples were subjected to rapid HPLC gel filtration which allowed us to separate IN from unfolded forms of RNase. The comparison of these samples, enriched in IN and depleted of unfolded forms, with unseparated control samples at the same stage of refolding allowed certain conclusions to be drawn concerning the properties of IN. First, the results show that the transition from IN to native RNase occurs with only small changes in fluorescence. This means that the major fluorescence changes seen during normal refolding experiments must be associated with changes in proline isomerization of unfolded species and/or with the refolding step itself but not with the IN----N step. Second, the fluorescence assay for isomerization of proline-93 shows that IN exists with proline-93 in a state of isomerization identical with or very similar to native RNase; i.e., proline-93 is cis in IN and not trans as suggested by others. All results are semiquantitatively consistent with our earlier refolding model and not nearly so consistent with alternative models which assume that most or all of the slow-refolding forms of RNase have proline-93 in the incorrect trans state.
为了确定在核糖核酸酶A重折叠过程中形成的类天然中间体（即IN）的结构特性，对重折叠样品进行了快速高效液相色谱凝胶过滤，这使我们能够将IN与核糖核酸酶的未折叠形式分离。将这些富含IN且未折叠形式减少的样品与处于相同重折叠阶段的未分离对照样品进行比较，从而得出关于IN特性的某些结论。首先，结果表明从IN到天然核糖核酸酶的转变仅伴随着荧光的微小变化。这意味着在正常重折叠实验中观察到的主要荧光变化必定与未折叠物种的脯氨酸异构化变化和/或与重折叠步骤本身相关，而不是与IN→N步骤相关。其次，对脯氨酸-93异构化的荧光测定表明，IN中的脯氨酸-93存在的异构化状态与天然核糖核酸酶相同或非常相似；即，脯氨酸-93在IN中是顺式的，而不是其他人所认为的反式。所有结果在半定量上与我们早期的重折叠模型一致，而与那些假设核糖核酸酶的大多数或所有慢重折叠形式的脯氨酸-93处于不正确反式状态的替代模型不太一致。
