Sagermann Martin, Baase Walter A, Mooers Blaine H M, Gay Leslie, Matthews Brian W
Institute of Molecular Biology, Howard Hughes Medical Institute, and Department of Physics, 1229, University of Oregon, Eugene, Oregon 97403-1229, USA.
Biochemistry. 2004 Feb 10;43(5):1296-301. doi: 10.1021/bi035702q.
In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.
在T4溶菌酶中，螺旋A位于序列的氨基末端，但在折叠结构中与C末端结构域相关联。为了研究这种排列对蛋白质折叠的影响，我们首先创建了一个环形排列变体，其新的氨基末端位于第12位残基处。实际上，这将对应于螺旋A的序列从分子的N末端移至C末端。该蛋白质与野生型非同源结晶，但具有非常相似的结构，表明由螺旋A和C末端结构域组成的单元可以从连续的多肽链中重构。该蛋白质比野生型稳定性差，但折叠速度稍快。然后我们产生了第二个变体，其中螺旋A序列附加在C末端（如在第一个变体中），但也在N末端恢复（如在野生型中）。这个变体有两个螺旋A序列，一个在N末端，另一个在C末端，每个都可以在折叠蛋白中竞争相同的位点。晶体结构表明，是N末端序列以与野生型相似的方式折叠，而C末端的拷贝被迫环出。这种蛋白质的稳定性更接近野生型，但其折叠速度明显较慢。折叠速度的降低归因于存在两个相同的序列片段，它们竞争单个相互排斥的位点。
