pH-Jump
Induced Leucine Zipper Folding beyond the
Diffusion Limit
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Abstract
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 (<i>o</i>-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 ∼1–2 μs time scale leading to a partially
folded α-helix even in the monomeric case and a second one leading
to the final coiled-coil structure on distinctively different time
scales of ∼30 μs for the cross-linked peptide and ∼200
μs 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