期刊
JOURNAL OF PHYSICAL CHEMISTRY B
卷 119, 期 4, 页码 1425-1432出版社
AMER CHEMICAL SOC
DOI: 10.1021/jp511539c
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资金
- Swiss National Foundation (SNP) [200021-119798]
- European Research Council (ERC) Advanced Investigator Grant (DYNALLO)
- Deutsche Forschungsgemeinschaft [SFB 969]
- Swiss National Science Foundation (SNF) [200021-119798] Funding Source: Swiss National Science Foundation (SNF)
The folding of a pH-sensitive leucine zipper, that is, a GCN4 mutant containing eight glutamic acid residues, has been investigated. A pH-jump induced by a caged proton (o-nitrobenzaldehyde, oNBA) is employed to initiate the process, and time-resolved IR spectroscopy of the amide I band is used to probe it. The experiment has been carefully designed to minimize the buffer capacity of the sample solution so that a large pH jump can be achieved, leading to a transition from a completely unfolded to a completely folded state with a single laser shot. In order to eliminate the otherwise rate-limiting diffusion-controlled step of the association of two peptides, they have been covalently linked. The results for the folding kinetics of the cross-linked peptide are compared with those of an unlinked peptide, which reveals a detailed picture of the folding mechanism. That is, folding occurs in two steps, one on an similar to 1-2 mu s time scale leading to a partially folded a-helix even in the monomeric case and a second one leading to the final coiled-coil structure on distinctively different time scales of similar to 30 mu s for the cross-linked peptide and similar to 200 mu s for the unlinked peptide. By varying the initial pH, it is found that the folding mechanism is consistent with a thermodynamic two-state model, despite the fact that a transient intermediate is observed in the kinetic experiment.
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