By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.

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With a combination of transient pump-probe IR spectroscopy and ab initio molecular dynamics, the controversial pico- and nanosecond steps of the o-nitrobenzaldehyde (oNBA) photoreaction have been investigated in aqueous solution. In this way, the measured reaction kinetics have been complemented with an atomistic picture of the reactive events as obtained with unbiased simulations in explicit solvent. Our results allow for a detailed description of the oNBA proton photorelease, a process of fundamental importance and relevant to the use of oNBA as a proton cage in many experiments. In a first step, a stable ketene intermediate is formed on a subpicosecond time scale. This intermediate reacts in a solvent assisted way with an OH transfer to produce nitrosobenzoic acid with a characteristic time of 7 ps. Finally, in permitting pH conditions, this product molecule dissociates a carboxyl proton with a 21 ns time constant. The particular combination of theory and experiment employed in this work appears to be sufficiently general and powerful to find widespread application in the study of ultrafast reactive systems.

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.

Acid–base systems are commonly expected to equilibrate on a time scale much faster than any other chemical reaction, so their composition can be deduced from the corresponding pK a or pK b values. In a pH-jump experiment done on a multi-acid/base pair system, it was found that it takes tens of microseconds before an equilibrium is established. Within that time, the system is kinetically driven, reaching surprising states very different from its final equilibrium; for example, carboxylate groups were protonated in the presence of hydroxyl ions.