title: pH-jump induced leucine zipper folding beyond the diffusion limit.
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.
authors.affiliation: Department of Chemistry, Universität Zürich , Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Mateusz L
authors.initials: ML
authors.lastname: Donten
authors.forename: Shabir
authors.initials: S
authors.lastname: Hassan
authors.forename: Alexander
authors.initials: A
authors.lastname: Popp
authors.forename: Jonathan
authors.initials: J
authors.lastname: Halter
authors.forename: Karin
authors.initials: K
authors.lastname: Hauser
authors.forename: Peter
authors.initials: P
authors.lastname: Hamm
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D004058:Diffusion; D006863:Hydrogen-Ion Concentration; D016350:Leucine Zippers; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides