Relocation or Duplication of the Helix A Sequence of T4 Lysozyme Causes Only Modest Changes in Structure but Can Increase or Decrease the Rate of Folding

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• 作者：Martin Sagermann ; Walter A. Baase ; Blaine H. M. Mooers ; Leslie Gay ; and Brian W. Matthews
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：February 10, 2004
• 年：2004
• 卷：43
• 期：5
• 页码：1296 - 1301
• 全文大小：379K
• 年卷期：v.43,no.5(February 10, 2004)
• ISSN：1520-4995

文摘

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associatedwith the C-terminal domain in the folded structure. To investigate the implications of this arrangementfor the folding of the protein, we first created a circularly permuted variant with a new amino terminusat residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminusof the molecule. The protein crystallized nonisomorphously with the wild type but has a very similarstructure, showing that the unit consisting of helix A and the C-terminal domain can be reconstitutedfrom 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 (asin the first variant), but was also restored at the N-terminus (as in the wild type). This variant has twohelix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete forthe same site in the folded protein. The crystal structure shows that it is the N-terminal sequence thatfolds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loopout. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantlyslower. The reduction in rate is attributed to the presence of the two identical sequence segments whichcompete for a single, mutually exclusive, site.