TEM-1 -Lactamase Folds in a Nonhierarchical Manner with Transient Non-Native Interactions Involving the C-Terminal Re
详细信息 查看全文
- 作者：Annabelle Lejeune ; Roger H. Pain ; Paulette Charlier ; Jean-Marie Frè ; re ; André ; Matagne
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：January 29, 2008
- 年：2008
- 卷：47
- 期：4
- 页码：1186 - 1193
- 全文大小：241K
- 年卷期：v.47,no.4(January 29, 2008)
- ISSN：1520-4995
文摘
The conformational stability and kinetics of refolding and unfolding of the W290F mutant ofTEM-1 
ars/beta2.gif" BORDER=0 ALIGN="middle">-lactamase have been determined as a function of guanidinium chloride concentration. The activityand spectroscopic properties of the mutant enzyme did not differ significantly from those of the wildtype, indicating that the mutation has only a very limited effect on the structure of the protein. The stabilityof the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globuleintermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminalregion of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seenin the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observedwith the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290.This residue is suggested to be buried in a nonpolar environment from which it has to escape duringsubsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in whichthe C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with anonhierarchical folding process.
ars/beta2.gif" BORDER=0 ALIGN="middle">-lactamase have been determined as a function of guanidinium chloride concentration. The activityand spectroscopic properties of the mutant enzyme did not differ significantly from those of the wildtype, indicating that the mutation has only a very limited effect on the structure of the protein. The stabilityof the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globuleintermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminalregion of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seenin the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observedwith the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290.This residue is suggested to be buried in a nonpolar environment from which it has to escape duringsubsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in whichthe C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with anonhierarchical folding process.