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 (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 ∼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.
Donten, Mateusz L