The Per-Arnt-Sim (PAS) domain is a ubiquitous protein module with a common three-dimensional fold involved in a wide range of regulatory and sensory functions in all domains of life. The activation of these functions is thought to involve partial unfolding of N- or C-terminal helices attached to the PAS domain. Here we use atomic force microscopy to probe receptor activation in single molecules of photoactive yellow protein (PYP), a prototype of the PAS domain family. Mechanical unfolding of Cys-linked PYP multimers in the presence and absence of illumination reveals that, in contrast to previous studies, the PAS domain itself is extended by ≈3 nm (at the 10-pN detection limit of the measurement) and destabilized by ≈30% in the light-activated state of PYP. Comparative measurements and steered molecular dynamics simulations of two double-Cys PYP mutants that probe different regions of the PAS domain quantify the anisotropy in stability and changes in local structure, thereby demonstrating the partial unfolding of their PAS domain upon activation. These results establish a generally applicable single-molecule approach for mapping functional conformational changes to selected regions of a protein. In addition, the results have profound implications for the molecular mechanism of PAS domain activation and indicate that stimulus-induced partial protein unfolding can be used as a signaling mechanism.

Optoelectronic properties of a polyphenylenevinylene-based oligomer and its paracylophane-linked dimer are studied using a variety of experimental and theoretical techniques. Despite the symmetrical structure and redshifted absorption of the dimer versus the monomer, an exciton picture is not the most appropriate. Electronic structure calculations establish changes in charge density upon optical excitation and show localized excitations that cannot be accounted for by a simple Frenkel exciton model. Visible frequency pump-probe anisotropy measurements suggest that the dimer should be considered as a three-level system with a fast, ∼130fs, internal conversion from the higher to lower energy excited electronic state. Signatures of nuclear relaxation processes are compared for electric field-resolved transient grating and two-dimensional photon echo spectra. These measurements reveal that nuclear relaxation occurs on similar time scales for the monomer and dimer. The connection between the spectral phase of four-wave mixing signals and the time dependent width of a nuclear wave packet is discussed. Semiempirical electronic structure and metropolis Monte Carlo calculations show that the dominant line broadening mechanisms for the monomer and dimer are associated with inter-ring torsional coordinates. Together, the theoretical calculations and electric field-resolved four-wave mixing experiments suggest that while the structure of dimer is more rigid than that of monomer, the difference in their rigidities is not sufficient to slow down excited state relaxation of dimer with respect to the monomer.

We present a comprehensive study that integrates experimental and theoretical nonequilibrium techniques to map energy landscapes along well defined pull-axis specific coordinates to elucidate mechanisms of protein unfolding. Single-molecule force-extension experiments along two different axes of photoactive yellow protein combined with nonequilibrium statistical mechanical analysis and atomistic simulation reveal energetic and mechanistic anisotropy. Steered molecular dynamics simulations and free-energy curves constructed from the experimental results reveal that unfolding along one axis exhibits a transition-state-like feature where six hydrogen bonds break simultaneously with weak interactions observed during further unfolding. The other axis exhibits a constant (unpeaked) force profile indicative of a noncooperative transition, with enthalpic (e.g., H-bond) interactions being broken throughout the unfolding process. Striking qualitative agreement was found between the force-extension curves derived from steered molecular dynamics calculations and the equilibrium free-energy curves obtained by Jarzynski-Hummer-Szabo analysis of the nonequilibrium work data. The anisotropy persists beyond pulling distances of more than twice the initial dimensions of the folded protein, indicating a rich energy landscape to the mechanically fully unfolded state. Our findings challenge the notion that cooperative unfolding is a universal feature in protein stability.