A Collapsed Intermediate with Nonnative Packing of Hydrophobic Residues in the Folding of TEM-1 -Lactamase
详细信息 查看全文
- 作者：Marc Vanhove ; Annabelle Lejeune ; Gilliane Guillaume ; Richard Virden ; Roger H. Pain ; Franz X. Schmid ; and Jean-Marie Frè ; re
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：February 17, 1998
- 年：1998
- 卷：37
- 期：7
- 页码：1941 - 1950
- 全文大小：181K
- 年卷期：v.37,no.7(February 17, 1998)
- ISSN：1520-4995
文摘
The kinetics of refolding of TEM-1 
-lactamase from solution inguanidine hydrochloridehave been investigated on the manual and stopped-flow mixing timescales. The kinetics of change offar-UV circular dichroism and of intrinsic and ANS fluorescence havebeen compared with changes inthe quenching of fluorescence by acrylamide as a probe of theaccessibility of solvent to tryptophan. Thebinding of ANS points to hydrophobic collapse in the very early stagesof folding which take place in theburst phase. This is accompanied by regain of 60-65% of nativeellipticity, indicating formation of asignificant proportion of secondary structure. Also in the burstphase, the tryptophan residues, which arelargely exposed to solvent in the native protein, become lessaccessible to acrylamide, and the intrinsicfluorescence increases markedly. An early intermediate is thusformed in which tryptophan is more buriedthan in the native protein. Further intermediates are formed overthe next 20 s. Quenching by acrylamideincreases during this period, as the transient nonnative state isdisrupted and the tryptophan residue(s)become(s) reexposed to solvent. The two slowest phases aredetermined by the isomerization of incorrectprolyl isomers, but double jump tryptophan fluorescence and acrylamidequenching experiments showlittle, if any, effect of proline isomerization on the earlier phases.Hydrophobic collapse thus occurs toa folding intermediate in which there is a nonnative element ofstructure which has to rearrange in thelater steps of folding, resulting in a nonhierarchical folding pathway.The C-terminal W290 is suggestedas being involved in the nonnative intermediate. -Lactamaseprovides further evidence for the occurrenceof nonnative intermediates in protein folding.
-lactamase from solution inguanidine hydrochloridehave been investigated on the manual and stopped-flow mixing timescales. The kinetics of change offar-UV circular dichroism and of intrinsic and ANS fluorescence havebeen compared with changes inthe quenching of fluorescence by acrylamide as a probe of theaccessibility of solvent to tryptophan. Thebinding of ANS points to hydrophobic collapse in the very early stagesof folding which take place in theburst phase. This is accompanied by regain of 60-65% of nativeellipticity, indicating formation of asignificant proportion of secondary structure. Also in the burstphase, the tryptophan residues, which arelargely exposed to solvent in the native protein, become lessaccessible to acrylamide, and the intrinsicfluorescence increases markedly. An early intermediate is thusformed in which tryptophan is more buriedthan in the native protein. Further intermediates are formed overthe next 20 s. Quenching by acrylamideincreases during this period, as the transient nonnative state isdisrupted and the tryptophan residue(s)become(s) reexposed to solvent. The two slowest phases aredetermined by the isomerization of incorrectprolyl isomers, but double jump tryptophan fluorescence and acrylamidequenching experiments showlittle, if any, effect of proline isomerization on the earlier phases.Hydrophobic collapse thus occurs toa folding intermediate in which there is a nonnative element ofstructure which has to rearrange in thelater steps of folding, resulting in a nonhierarchical folding pathway.The C-terminal W290 is suggestedas being involved in the nonnative intermediate. -Lactamaseprovides further evidence for the occurrenceof nonnative intermediates in protein folding.